RU2833504C1 - Pharmaceutical composition containing double-stranded ribonucleic acid inhibiting expression of complement component c5 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a pharmaceutical composition for inhibiting expression of complement component C5, containing a lipid complex, where the lipid complex includes double-stranded ribonucleic acid, and the pH of the solution of the lipid complex is 2.0 or more and 5.0 or less, or 7.5 or more and 11.0 or less. Also disclosed is a method of producing a pharmaceutical composition and a method of stabilizing it. Invention also relates to a method of treating paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.

EFFECT: invention provides effective suppression of expression of complement component C5.

23 cl, 6 dwg, 31 tbl, 16 ex

Description

[Field of technology]

[0001] The present invention relates to a pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) capable of suppressing the expression of complement component C5. More particularly, the present invention relates to a pharmaceutical composition comprising a lipid complex comprising a double-stranded ribonucleic acid capable of suppressing the expression of complement component C5, a method for producing the pharmaceutical composition, and a method for stabilizing the pharmaceutical composition .

[State of the art]

[0002] The group of proteins called the complement system includes proteins designated C1-C9, and these proteins are sequentially activated in three different pathways (classical pathway, lectin pathway, alternative pathway) to develop an immune response. The fifth component of complement, C5, is cleaved into C5a and C5b by C5 convertase. C5a is called an anaphylatoxin and induces an inflammatory response in various cells via C5aR (CD88) and C5L2 (GPR77). C5b sequentially reacts with C6-C9, ultimately forming the membrane attack complex (MAC), which causes bacteriolysis of pathogens or cell lysis. If the complement system is not properly controlled or is overly activated, the complement system can cause severe cytotoxicity to host cells.

[0003] Based on previous studies, complement component C5 is associated with various diseases, including paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), myasthenia gravis (MG), neuromyelitis optica (NMO), antibody-mediated rejection in renal transplantation, Guillain-Barré syndrome, anti-neutrophil cytoplasmic antibody-associated vasculitis (ANCA-associated vasculitis), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), autoimmune encephalitis, IgG4-related diseases, asthma, antiphospholipid antibody syndrome, ischemia-reperfusion injury, typical hemolytic uremic syndrome (tHUS), multifocal motor neuropathy (MMN), multiple sclerosis (MS), thrombotic thrombocytopenic purpura (TTP), spontaneous abortion, recurrent miscarriage, traumatic brain injury, cold agglutinin disease, dermatomyositis, Shiga toxin-producing Escherichia coli (E. coli)-associated hemolytic uremic syndrome, graft dysfunction, myocardial infarction, sepsis, atherosclerosis, septic shock, spinal cord injury, psoriasis, autoimmune hemolytic anemia (AIHA), antiphospholipid syndrome (APS), myocarditis, hemorrhagic vasculitis, Takayasu disease, and Kawasaki disease (arteritis). Thus, inhibition or suppression of the expression of complement component C5 is expected to lead to successful treatment of these diseases. In particular, inhibition of complement component C5 is suggested to be effective for the treatment or prevention of paroxysmal nocturnal hemoglobinuria (non-patent literature 1), atypical hemolytic uremic syndrome (non-patent literature 2), severe myasthenia gravis (non-patent literature 3), neuromyelitis optica (non-patent literature 4), and antibody-mediated renal transplant rejection (non-patent literature 5).

[0004] The anti-C5 monoclonal antibody, eculizumab (Soliris (registered trade name)), exhibits high affinity for the complement component C5 and suppresses excessive activation of the complement system by inhibiting the cleavage of C5 into C5a/C5b and the concomitant formation of the membrane attack complex. Therefore, eculizumab exhibits an inhibitory effect on hemolysis and is thus known as a therapeutic agent for use in paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. In addition, eculizumab is known as a therapeutic agent for use in generalized myasthenia gravis (gMG). However, eculizumab is very expensive, and therefore, the development of alternative agents applicable to the treatment and prevention of complement component C5-mediated diseases is needed.

[0005] Examples of methods for suppressing the expression of complement component C5 include methods that use RNA interference (also referred to hereinafter as "RNAi"). For example, a double-stranded ribonucleic acid (dsRNA)-based agent that induces cleavage of an RNA transcript of the C5 component gene via an RNA-induced silencing complex (RISC) is known (Patent Literature 1).

[List of references]

[Patent Literature]

[0006] [Patent Literature 1] WO 2014/160129

[Non-patent literature sources]

[0007] [Non-patent literature 1] Non-patent literature 1: Peter Hillmen et al., The New England Journal of Medicine 2004 Feb 5; 350(6): 552-559.

[Non-patent literature source 2] Legendre C.M. et al., The New England Journal of Medicine 2013 Jun 6; 368(23): 2169-2181.

[Non-patent literature 3] Howard J.F. Jr et al., Muscle Nerve 2013 Jul; 48(1): 76-84.

[Non-patent literature source 4] Pittock S.J. et al., The Lancet Neurology 2013 Jun; 12(6): 554-562.

[Non-patent literature 5] Stegall M.D. et al., American Journal of Transplantation 2011 Nov; 11(11): 2405-2413.

[Essence of the invention]

[Technical task]

[0008] The aim of the present invention is to obtain a new pharmaceutical composition containing a lipid complex containing double-stranded ribonucleic acid for suppressing the expression of complement component C5, a method for producing the pharmaceutical composition and a method for stabilizing the pharmaceutical composition .

[Problem solution]

[0009] The present invention provides, for example, the following items <1> to <81>.

<1> A pharmaceutical composition containing

lipid complex,

wherein the lipid complex comprises a double-stranded ribonucleic acid containing a combination of a sense strand and an antisense strand,

the combination of the sense strand and the antisense strand is selected from the group consisting of a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 159 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 160, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 141 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 142, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 143 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 144, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 145 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 146, a sense strand consisting of the nucleotide sequence presented under SEQ ID NO: 147 and an antisense strand consisting of the nucleotide sequence presented under SEQ ID NO: 148, as well as a sense strand consisting of the nucleotide sequence presented under SEQ ID NO: 153 and an antisense strand consisting of the nucleotide sequence presented under SEQ ID NO: 154, and

The pH of the lipid complex solution is 5.0 or less or 7.5 or greater.

<2> A pharmaceutical composition containing

lipid complex,

wherein the lipid complex comprises a double-stranded ribonucleic acid comprising a sense strand consisting of the nucleotide sequence presented under SEQ ID NO: 145 and an antisense strand consisting of the nucleotide sequence presented under SEQ ID NO: 146, and

The pH of the lipid complex solution is 5.0 or less or 7.5 or greater.

<3> The pharmaceutical composition according to <1> or <2>, wherein the pH of the lipid complex solution is 2.0 or greater and 5.0 or less, or 7.5 or greater and 11.0 or less.

<4> The pharmaceutical composition according to <1> or <2>, wherein the pH of the lipid complex solution is 5.0 or less.

<5> The pharmaceutical composition according to <1> or <2>, wherein the pH of the lipid complex solution is 2.0 or greater and 5.0 or less.

<6> The pharmaceutical composition according to <1> or <2>, wherein the pH of the lipid complex solution is 7.5 or greater.

<7> The pharmaceutical composition according to <1> or <2>, wherein the pH of the lipid complex solution is 7.5 or greater and 11.0 or less.

<8> The pharmaceutical composition according to <1> or <2>, wherein the pH of the lipid complex solution is 7.5 or greater and 10.0 or less.

<9> The pharmaceutical composition according to <1> or <2>, wherein the pH of the lipid complex solution is 7.5 or greater and 9.5 or less.

<10> The pharmaceutical composition according to <1> or <2>, wherein the pH of the lipid complex solution is 7.5 or greater and 9.0 or less.

<11> The pharmaceutical composition according to <1> or <2>, wherein the pH of the lipid complex solution is 7.5 or greater and 8.5 or less.

<12> The pharmaceutical composition according to any one of <1> to <11>, wherein the average particle size of the lipid complex is 100 nm or less.

<13> The pharmaceutical composition according to any one of <1> to <12>, wherein the average particle size of the lipid complex is 65 nm or more and 100 nm or less.

<14> The pharmaceutical composition according to any one of <1> to <12>, wherein the average particle size of the lipid complex is 80 nm or more and 100 nm or less.

<15> The pharmaceutical composition according to any one of <1> to <12>, wherein the average particle size of the lipid complex is 85 nm or more and 100 nm or less.

<16> The pharmaceutical composition according to any one of <1>-<15>, wherein the change in the average particle size of the lipid complex after storage for 2 weeks is 10% or less of the average particle size of the lipid complex before storage.

<17> The pharmaceutical composition according to any one of <1>-<15>, wherein the change in the average particle size of the lipid complex after storage for 2 weeks is 8% or less of the average particle size of the lipid complex before storage.

<18> The pharmaceutical composition according to any one of <1>-<15>, wherein the change in the average particle size of the lipid complex after storage for 2 weeks is 5% or less of the average particle size of the lipid complex before storage.

<19> The pharmaceutical composition according to any one of <1>-<15>, wherein the change in the average particle size of the lipid complex after storage for 1 month is 10% or less of the average particle size of the lipid complex before storage.

<20> The pharmaceutical composition according to any one of <1> to <15>, wherein the change in the average particle size of the lipid complex after storage for 1 month is 8% or less of the average particle size of the lipid complex before storage.

<21> The pharmaceutical composition according to any one of <1>-<15>, wherein the change in the average particle size of the lipid complex after storage for 1 month is 5% or less of the average particle size of the lipid complex before storage.

<22> The pharmaceutical composition according to any one of <1> to <15>, wherein the change in the average particle size of the lipid complex after storage for 3 months is 10% or less of the average particle size of the lipid complex before storage.

<23> The pharmaceutical composition according to any one of <1> to <15>, wherein the change in the average particle size of the lipid complex after storage for 3 months is 8% or less of the average particle size of the lipid complex before storage.

<24> The pharmaceutical composition according to any one of <1>-<15>, wherein the change in the average particle size of the lipid complex after storage for 3 months is 5% or less of the average particle size of the lipid complex before storage.

<25> A pharmaceutical composition according to any one of <16>-<24>, wherein the change in average particle size is an increase in average particle size.

<26> A pharmaceutical composition according to any one of <16>-<25>, wherein the storage conditions of the pharmaceutical composition are 2-8°C.

<27> A pharmaceutical composition according to any one of <16>-<25>, wherein the storage conditions of the pharmaceutical composition are 25°C.

<28> A pharmaceutical composition according to any one of <1>-<27>, wherein the lipid complex comprises

cationic lipid and

at least one lipid selected from the group consisting of a neutral lipid, a polyethyleneglycol-modified lipid, and a sterol.

<29> A pharmaceutical composition according to any one of <1>-<28>, wherein the lipid complex comprises a cationic lipid, a neutral lipid, a polyethyleneglycol-modified lipid and a sterol.

<30> The pharmaceutical composition according to <28> or <29>, wherein the cationic lipid is selected from the group consisting of 1-oxo-1-(undecan-5-yloxy)nonadecan-10-yl-1-methylpiperidine-4-carboxylate, 1-((2-butyloctyl)oxy)-1-oxononadecane-10-yl-1-methylpiperidine-4-carboxylate, 1-oxo-1-(undecan-5-yloxy)heptadecan-8-yl-1-methylpiperidine 4-carboxylate, 21-oxo-21-(undecan-5-yloxy)henicosan-10-yl-1-methylpiperidine 4-carboxylate, 21-(octan-3-yloxy)-21-oxogenicosan-10-yl-1-methylpiperidine-4-carboxylate, 1-((2-butyloctyl)oxy)-1-oxoicosan-10-yl-1-methylpiperidine-4-carboxylate, (Z)-1-((2-butylnon-3-en-1-yl)oxy)-1-oxoicosan-10-yl-1-methylpiperidine-4-carboxylate, 1-oxo-1-((3-pentyloctyl)oxy)icosan-10-yl-1-methylpiperidine-4-carboxylate, 1-((3,4-dipropylheptyl)oxy)-1-oxoicosan-10-yl-1-methylpiperidine-4-carboxylate, 1-((6-(butyldisulfanyl)-3-(3-(butyldisulfanyl)propyl)hexyl)oxy)-1-oxoicosan-10-yl-1-methylpiperidin-4-carboxylate, 2-butyloctyl-10-((4-(dimethylamino)butanoyl)oxy)icosanoate, 2-{9-[(2-butyloctyl)oxy]-9-oxononyl}dodecyl-1-methylpiperidine-4-carboxylate, 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl-1-methylpiperidine-4-carboxylate, 2-nonyl-11-oxo-11-[(3-pentyloctyl)oxy]undecyl-1-methylpiperidine-4-carboxylate, bis(3-pentyloctyl)-9-{[(1-methylpiperidin-4-carbonyl)oxy]methyl}heptadecanedioate, di[(Z)-2-nonen-1-yl]-9-{[(1-methylpiperidin-4-carbonyl)oxy]methyl}heptadecanedioate, 1-(2-octylcyclopropyl)heptadecan-8-yl-1-methylpiperidine-4-carboxylate, (3S)-2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl-1-methylpyrrolidine-3-carboxylate and (3R)-2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl-1-methylpyrrolidine-3-carboxylate.

<31> The pharmaceutical composition according to any one of <28> to <30>, wherein the cationic lipid is selected from the group consisting of 1-((2-butyloctyl)oxy)-1-oxoicosan-10-yl-1-methylpiperidine-4-carboxylate, 1-((2-butyloctyl)oxy)-1-oxononadecane-10-yl-1-methylpiperidine-4-carboxylate, 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl-1-methylpiperidine-4-carboxylate, 1-(2-octylcyclopropyl)heptadecan-8-yl-1-methylpiperidine-4-carboxylate, (3S)-2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpyrrolidine-3-carboxylate and (3R)-2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpyrrolidine-3-carboxylate.

<32> A pharmaceutical composition according to any one of <28>-<31>, wherein the cationic lipid is 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl-1-methylpiperidine-4-carboxylate.

<33> The pharmaceutical composition according to any one of <28>-<32>, wherein the neutral lipid is a phospholipid or ceramide.

<34> The pharmaceutical composition according to <33>, wherein the phospholipid is selected from the group consisting of DOPE, POPE, HSPC, SOPC, POPC, EPC, DMPC, DPPC, DSPC, DAPC, DBPC, DLPC, DOPC, DOPG, DPPG, DSPG, DOPS, DOPE-MAL and sphingomyelin.

<35> The pharmaceutical composition according to <33> or <34>, wherein the phospholipid is selected from the group consisting of DOPE, HSPC, DPPC, DSPC and DAPC.

<36> The pharmaceutical composition according to any one of <33>-<35>, wherein the phospholipid is DSPC.

<37> The pharmaceutical composition according to any one of <28>-<36>, wherein the polyethyleneglycol-modified lipid is selected from the group consisting of PEG2000-DMG, PEG2000-DPG, PEG2000-DSG, PEG5000-DMG, PEG5000-DPG, PEG5000-DSG, PEG-cDMA, PEG-C-DOMG, PEG-DAG, PEG-DAA, PEG-phospholipid, PEG-cholesterol and PEG-ceramide (Cer).

<38> A pharmaceutical composition according to any one of <28>-<37>, wherein the polyethyleneglycol-modified lipid is selected from the group consisting of PEG2000-DMG, PEG2000-DPG, PEG2000-DSG, PEG-cDMA and PEG-C-DOMG.

<39> The pharmaceutical composition according to any one of <28>-<38>, wherein the polyethyleneglycol-modified lipid is PEG2000-DMG.

<40> A pharmaceutical composition according to any one of <28>-<39>, wherein the sterol is selected from the group consisting of cholesterol, dihydrocholesterol, lanosterol, β-sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, fucosterol and 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol).

<41> A pharmaceutical composition according to any one of <28>-<40>, wherein the sterol is selected from the group consisting of cholesterol, dihydrocholesterol, lanosterol and β-sitosterol.

<42> A pharmaceutical composition according to any one of <28>-<41>, wherein the sterol is cholesterol.

<43> A pharmaceutical composition according to any one of <1>-<42>, wherein the lipid complex comprises 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl-1-methylpiperidine-4-carboxylate, DSPC, PEG2000-DMG and cholesterol.

<44> A pharmaceutical composition according to any one of <1>-<43>, wherein the molar ratio of cationic lipid/neutral lipid/polyethyleneglycol-modified lipid/sterol in the lipid complex is 30-90/0.1-20/0.01-10/0.1-70.

<45> A pharmaceutical composition according to any one of <1>-<43>, wherein the molar ratio of cationic lipid/neutral lipid/polyethyleneglycol-modified lipid/sterol in the lipid complex is 40-70/3-15/0.1-3/15-60.

<46> A pharmaceutical composition according to any one of <1>-<43>, wherein the molar ratio of cationic lipid/neutral lipid/polyethyleneglycol-modified lipid/sterol in the lipid complex is 60/10.5/1.5/28.

<47> A pharmaceutical composition according to any one of <1>-<46>, wherein the lipid complex is a lipid nanoparticle (LNP).

<48> A pharmaceutical composition according to any one of <1>-<47>, wherein the lipid complex encapsulates a double-stranded ribonucleic acid comprising a combination of a sense strand and an antisense strand.

<49> A pharmaceutical composition according to any one of <1>-<48>, further comprising a pharmaceutically acceptable carrier.

<50> A pharmaceutical composition according to any one of <1>-<49> for the treatment of paroxysmal nocturnal hemoglobinuria.

<51> A pharmaceutical composition according to any one of <1>-<49> for the treatment of atypical hemolytic uremic syndrome.

<52> A method for treating paroxysmal nocturnal hemoglobinuria, which involves

administration of the pharmaceutical composition according to any of <1>-<49> to a patient in need thereof.

<53> A method for treating atypical hemolytic uremic syndrome, which includes

administration of the pharmaceutical composition according to any of <1>-<49> to a patient in need thereof.

<54> A pharmaceutical composition according to any one of <1>-<49> for use in the treatment of paroxysmal nocturnal hemoglobinuria.

<55> A pharmaceutical composition according to any of <1>-<49> for use in the treatment of atypical hemolytic uremic syndrome.

<56> A method for producing a pharmaceutical composition according to any of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 5.0 or less or 7.5 or more.

<57> The method according to <56> for producing a pharmaceutical composition according to any of <1>-<55>, comprising

adjusting the pH of the lipid complex solution to 2.0 or greater and 5.0 or less, or 7.5 or greater and 11.0 or less.

<58> The method according to <56> for producing a pharmaceutical composition according to any of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 5.0 or less.

<59> The method according to <56> for producing a pharmaceutical composition according to any of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 2.0 or greater and 5.0 or less.

<60> The method according to <56> for producing a pharmaceutical composition according to any of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 7.5 or higher.

<61> The method according to <56> for producing a pharmaceutical composition according to any of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 7.5 or greater and 11.0 or less.

<62> The method according to <56> for producing a pharmaceutical composition according to any of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 7.5 or greater and 10.0 or less.

<63> The method according to <56> for producing a pharmaceutical composition according to any of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 7.5 or greater and 9.5 or less.

<64> The method according to <56> for producing a pharmaceutical composition according to any of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 7.5 or greater and 9.0 or less.

<65> The method according to <56> for producing a pharmaceutical composition according to any of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 7.5 or greater and 8.5 or less.

<66> A method according to any of <56> to <65> for producing a pharmaceutical composition according to any of <1> to <55>, comprising

mixing an organic solvent containing (I) a cationic lipid and (II) at least one lipid selected from the group consisting of a neutral lipid, a polyethyleneglycol-modified lipid, and a sterol, and an aqueous solution containing a double-stranded ribonucleic acid containing a combination of a sense strand and an antisense strand, to obtain a mixed solution.

<67> The method according to <66> for producing a pharmaceutical composition according to any of <1>-<55>, further comprising

removal of organic solvent from a mixed solution.

<68> A method for stabilizing a pharmaceutical composition according to any of <1>-<55>, comprising:

bringing the pH of the lipid complex solution to 5.0 or less or 7.5 or more.

<69> The method according to <68> for stabilizing a pharmaceutical composition according to any of <1>-<55>, comprising

adjusting the pH of the lipid complex solution to 2.0 or greater and 5.0 or less, or 7.5 or greater and 11.0 or less.

<70> The method according to <68> for stabilizing a pharmaceutical composition according to any one of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 5.0 or less.

<71> The method according to <68> for stabilizing a pharmaceutical composition according to any one of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 2.0 or greater and 5.0 or less.

<72> The method according to <68> for stabilizing a pharmaceutical composition according to any of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 7.5 or higher.

<73> The method according to <68> for stabilizing a pharmaceutical composition according to any of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 7.5 or greater and 11.0 or less.

<74> The method according to <68> for stabilizing a pharmaceutical composition according to any one of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 7.5 or greater and 10.0 or less.

<75> The method according to <68> for stabilizing a pharmaceutical composition according to any one of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 7.5 or greater and 9.5 or less.

<76> The method according to <68> for stabilizing a pharmaceutical composition according to any of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 7.5 or greater and 9.0 or less.

<77> The method according to <68> for stabilizing a pharmaceutical composition according to any of <1>-<55>, comprising

bringing the pH of the lipid complex solution to 7.5 or greater and 8.5 or less.

<78> A method according to any of <68> to <77> for stabilizing a pharmaceutical composition according to any of <1> to <55>, comprising

mixing an organic solvent containing (I) a cationic lipid and (II) at least one lipid selected from the group consisting of a neutral lipid, a polyethyleneglycol-modified lipid, and a sterol, and an aqueous solution containing a double-stranded ribonucleic acid containing a combination of a sense strand and an antisense strand, to obtain a mixed solution.

<79> The method of <78> for stabilizing a pharmaceutical composition according to any one of <1>-<55>, further comprising

removal of organic solvent from a mixed solution.

<80> The method according to any one of <68> to <79> for stabilizing the pharmaceutical composition according to any one of <1> to <55>, wherein the method of stabilizing the pharmaceutical composition is a method of suppressing a change in the average particle size of a lipid complex in the pharmaceutical composition.

<81> The method according to <80> for stabilizing the pharmaceutical composition according to any one of <1> to <55>, wherein the method of suppressing a change in the average particle size is a method of suppressing an increase in the average particle size.

[Beneficial effects of the invention]

[0010] According to the present invention, a new pharmaceutical composition may be provided, comprising a double-stranded ribonucleic acid capable of suppressing the expression of complement component C5, a method for producing the pharmaceutical composition, and a method for stabilizing the pharmaceutical composition.

[0011] The pharmaceutical composition of the present invention can suppress the expression of complement component C5 to suppress hemolysis, and therefore can be used as a therapeutic agent for paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS).

[Brief description of graphic materials]

[0012]

[Fig. 1]

Fig. 1 shows graphs representing the results of assessing the residual levels of C5 component mRNA in the liver after administration of siRNA-008 and the residual levels of C5 component mRNA in the liver after administration of siRNA-008-34 in Example 5.

[Fig. 2]

Fig. 2 shows graphs representing the results of evaluating the residual levels of the C5 component in blood plasma after administration of siRNA-008 and the residual levels of the C5 component in blood plasma after administration of siRNA-008-34 in Example 5.

[Fig. 3]

Fig. 3 shows a graph representing the results of the evaluation of the activity of the complement system after the administration of siRNA-008-34 in Example 8.

[Fig. 4]

Fig. 4 shows a graph representing the results of the evaluation of the activity of the complement system after the administration of siRNA-008-34 in Example 9.

[Fig. 5]

Fig. 5 shows a graph representing the results of the evaluation of the activity of the complement system after the administration of siRNA-008-34 in Example 10.

[Fig. 6]

Fig. 6 shows a graph representing the results of the evaluation of the activity of the complement system after the administration of siRNA-008-34 in Example 11.

[Description of embodiments]

[0013] Examples of genes encoding the complement component C5 that are targeted by the double-stranded ribonucleic acid of an embodiment include, but are not limited to, C5 component genes obtained from humans, mice, and monkeys. Sequence information for the C5 component gene is available from publicly available databases, including sequence information filed in a registry such as GenBank provided by the National Center for Biotechnology Information (NCBI), or it can be obtained by designing a primer based on the nucleotide sequence information of the C5 component gene from a closely related animal species and then cloning it with RNA isolated from the desired animal species. Examples of an mRNA transcript sequence corresponding to a human C5 component target gene include the human C5 component mRNA transcript sequence filed under GenBank Accession No. NM_001735.2 (GI: 38016946). The term "C5 component gene" as used herein is not limited to a gene having a specific sequence. For example, naturally occurring C5 component genes with single nucleotide polymorphisms may also fall within the definition of this term.

[0014] In an embodiment of a double-stranded ribonucleic acid comprising a sense strand and an antisense strand, the combination of the sense strand and the antisense strand is selected from the group consisting of a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 13 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 14, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 159 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 160, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 115 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 116, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 117, and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 118, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 119 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 120, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 121 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 122, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 123 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 124, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 125 and an antisense strand consisting of a nucleotide sequence as set forth in SEQ ID NO: 126, a sense strand consisting of the nucleotide sequence as set forth in SEQ ID NO: 127 and an antisense strand consisting of the nucleotide sequence as set forth in SEQ ID NO: 128, a sense strand consisting of the nucleotide sequence as set forth in SEQ ID NO: 129 and an antisense strand consisting of the nucleotide sequence as set forth in SEQ ID NO: 130, a sense strand consisting of the nucleotide sequence as set forth in SEQ ID NO: 131 and an antisense strand consisting of the nucleotide sequence as set forth in SEQ ID NO: 132, a sense strand consisting of the nucleotide sequence as set forth in SEQ ID NO: 133 and an antisense strand consisting of the nucleotide sequence as set forth in SEQ ID NO: 134, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 137 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 138, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 139 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 140, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 141 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 142, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 143 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 144, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 145, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 146, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 147, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 148, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 149, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 150, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 151, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 152, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 153, and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 154, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 155, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 156, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 157, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 158, a sense strand consisting of the nucleotide sequence shown the sequence presented by SEQ ID NO: 145 and an antisense strand consisting of the nucleotide sequence presented by SEQ ID NO: 146, a sense strand consisting of the nucleotide sequence presented by SEQ ID NO: 147 and an antisense strand consisting of the nucleotide sequence presented by SEQ ID NO: 148, a sense strand consisting of the nucleotide sequence presented by SEQ ID NO: 149 and an antisense strand consisting of the nucleotide sequence presented by SEQ ID NO: 150, a sense strand consisting of the nucleotide sequence presented by SEQ ID NO: 151 and an antisense strand consisting of the nucleotide sequence presented by SEQ ID NO: 152, and a sense strand consisting of the nucleotide sequence presented by SEQ ID NO: 153, and an antisense strand consisting of the nucleotide sequence presented under SEQ ID NO: 154. These combinations correspond to the sequences siRNA-008, siRNA-008-01, siRNA-008-02, siRNA-008-08, siRNA-008-09, siRNA-008-10, siRNA-008-11, siRNA-008-12, siRNA-008-13, siRNA-008-14, siRNA-008-22, siRNA-008-23, siRNA-008-30, siRNA-008-31, siRNA-008-32, siRNA-008-33, siRNA-008-34, siRNA-008-35, siRNA-008-36, siRNA-008-37 and siRNA-008-38, respectively, in the present description.

[0015] In an embodiment of a double-stranded ribonucleic acid, the sense strand and the antisense strand form a pair in the form of any of the combinations (1) - (21):

(1) a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 13 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 14;

(2) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 159 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 160;

(3) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 115 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 116;

(4) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 117 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 118;

(5) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 119 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 120;

(6) a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 121 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 122;

(7) a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 123 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 124;

(8) a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 125 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 126;

(9) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 127 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 128;

(10) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 129 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 130;

(11) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 131 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 132;

(12) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 133 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 134;

(13) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 137 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 138;

(14) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 139 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 140;

(15) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 141 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 142;

(16) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 143 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 144;

(17) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 145 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 146;

(18) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 147 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 148;

(19) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 149 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 150;

(20) a sense strand consisting of the nucleotide sequence presented as SEQ ID NO: 151 and an antisense strand consisting of the nucleotide sequence presented as SEQ ID NO: 152;

(21) a sense strand consisting of the nucleotide sequence presented under SEQ ID NO: 153, and an antisense strand consisting of the nucleotide sequence presented under SEQ ID NO: 154.

[0016] Each of the combinations (1) to (21) of the sense strand and the antisense strand includes a region of complementarity between the strands. For example, a double-stranded ribonucleic acid including a combination of (1) the sense strand shown as SEQ ID NO: 13 and the antisense strand shown as SEQ ID NO: 14 includes the following complementary strands (the dT^dT pair at the 3' end is not shown, see Table 1 for more details):

5'-uGGuAuAuGuGuuGcuGAu-3' (SEQ ID NO: 13)

3'-AcCAuAuAcAcAAcGAcUA-5' (SEQ ID NO: 14).

[0017] In an embodiment of a double-stranded ribonucleic acid comprising a sense strand and an antisense strand, the combination of the sense strand and the antisense strand is selected from the group consisting of a sense strand consisting of a nucleotide sequence as set forth in SEQ ID NO: 159 and an antisense strand consisting of a nucleotide sequence as set forth in SEQ ID NO: 160, a sense strand consisting of a nucleotide sequence as set forth in SEQ ID NO: 139 and an antisense strand consisting of a nucleotide sequence as set forth in SEQ ID NO: 140, a sense strand consisting of a nucleotide sequence as set forth in SEQ ID NO: 141 and an antisense strand consisting of a nucleotide sequence as set forth in SEQ ID NO: 142, a sense strand consisting of a nucleotide sequence as set forth in SEQ ID NO: ID NO: 143, and an antisense strand consisting of the nucleotide sequence presented by SEQ ID NO: 144, a sense strand consisting of the nucleotide sequence presented by SEQ ID NO: 145 and an antisense strand consisting of the nucleotide sequence presented by SEQ ID NO: 146, a sense strand consisting of the nucleotide sequence presented by SEQ ID NO: 147 and an antisense strand consisting of the nucleotide sequence presented by SEQ ID NO: 148, and a sense strand consisting of the nucleotide sequence presented by SEQ ID NO: 153 and an antisense strand consisting of the nucleotide sequence presented by SEQ ID NO: 154. The combinations correspond to the sequences siRNA-008-01, siRNA-008-31, siRNA-008-32, siRNA-008-33, siRNA-008-34, siRNA-008-35 and siRNA-008-38, respectively, in the present description.

[0018] In an embodiment of a double-stranded ribonucleic acid comprising a sense strand and an antisense strand, the combination of the sense strand and the antisense strand is selected from the group consisting of a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 159 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 160, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 141 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 142, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: 143 and an antisense strand consisting of the nucleotide sequence shown as SEQ ID NO: 144, a sense strand consisting of the nucleotide sequence shown as SEQ ID NO: ID NO: 145, and an antisense strand consisting of the nucleotide sequence presented by SEQ ID NO: 146, a sense strand consisting of the nucleotide sequence presented by SEQ ID NO: 147 and an antisense strand consisting of the nucleotide sequence presented by SEQ ID NO: 148, as well as a sense strand consisting of the nucleotide sequence presented by SEQ ID NO: 153 and an antisense strand consisting of the nucleotide sequence presented by SEQ ID NO: 154. The combinations correspond to the sequences siRNA-008-01, siRNA-008-32, siRNA-008-33, siRNA-008-34, siRNA-008-35 and siRNA-008-38, respectively, in the present description.

[0019] In an embodiment of a double-stranded ribonucleic acid comprising a sense strand and an antisense strand, the combination of the sense strand and the antisense strand is selected from the group consisting of a sense strand consisting of a nucleotide sequence as set forth in SEQ ID NO: 141 and an antisense strand consisting of a nucleotide sequence as set forth in SEQ ID NO: 142, a sense strand consisting of a nucleotide sequence as set forth in SEQ ID NO: 143 and an antisense strand consisting of a nucleotide sequence as set forth in SEQ ID NO: 144, a sense strand consisting of a nucleotide sequence as set forth in SEQ ID NO: 145 and an antisense strand consisting of a nucleotide sequence as set forth in SEQ ID NO: 146, a sense strand consisting of a nucleotide sequence as set forth in SEQ ID NO: ID NO: 147, and an antisense strand consisting of the nucleotide sequence presented under SEQ ID NO: 148, as well as a sense strand consisting of the nucleotide sequence presented under SEQ ID NO: 153, and an antisense strand consisting of the nucleotide sequence presented under SEQ ID NO: 154. The combinations correspond to the sequences siRNA-008-32, siRNA-008-33, siRNA-008-34, siRNA-008-35 and siRNA-008-38, respectively, in the present description.

[0020] In an embodiment, a double-stranded ribonucleic acid comprising a sense strand and an antisense strand comprises a sense strand consisting of the nucleotide sequence set forth under SEQ ID NO: 159 and an antisense strand consisting of the nucleotide sequence set forth under SEQ ID NO: 160. The combination corresponds to the sequence of siRNA-008-01 herein.

[0021] In an embodiment, a double-stranded ribonucleic acid comprising a sense strand and an antisense strand comprises a sense strand consisting of the nucleotide sequence set forth in SEQ ID NO: 141 and an antisense strand consisting of the nucleotide sequence set forth in SEQ ID NO: 142. The combination corresponds to the sequence of siRNA-008-32 herein.

[0022] In an embodiment, a double-stranded ribonucleic acid comprising a sense strand and an antisense strand comprises a sense strand consisting of the nucleotide sequence set forth in SEQ ID NO: 143 and an antisense strand consisting of the nucleotide sequence set forth in SEQ ID NO: 144. The combination corresponds to the sequence of siRNA-008-33 herein.

[0023] In an embodiment, a double-stranded ribonucleic acid comprising a sense strand and an antisense strand comprises a sense strand consisting of the nucleotide sequence set forth in SEQ ID NO: 145 and an antisense strand consisting of the nucleotide sequence set forth in SEQ ID NO: 146. The combination corresponds to the sequence of siRNA-008-34 herein.

[0024] In an embodiment, a double-stranded ribonucleic acid comprising a sense strand and an antisense strand comprises a sense strand consisting of the nucleotide sequence set forth in SEQ ID NO: 147 and an antisense strand consisting of the nucleotide sequence set forth in SEQ ID NO: 148. The combination corresponds to the sequence of siRNA-008-35 herein.

[0025] In an embodiment, a double-stranded ribonucleic acid comprising a sense strand and an antisense strand comprises a sense strand consisting of the nucleotide sequence set forth in SEQ ID NO: 153 and an antisense strand consisting of the nucleotide sequence set forth in SEQ ID NO: 154. The combination corresponds to the sequence of siRNA-008-38 herein.

[0026] In an embodiment, the antisense strand is substantially complementary to at least a portion of the mRNA transcript of the C5 component gene. As used herein, the phrase "substantially complementary" includes not only instances where the antisense strand is completely complementary to a portion of the mRNA transcript of the C5 component gene, but also instances where there are one to more acceptable mismatches between the antisense strand and the portion of the mRNA transcript of the C5 component gene.

[0027] In an embodiment, the sense strand is substantially complementary to at least a portion of the nucleotide sequence of the antisense strand. The phrase "substantially complementary" includes not only cases where the sense strand is completely complementary to a portion of the nucleotide sequence of the antisense strand, but also cases where there are one to more acceptable mismatches between the sense strand and a portion of the nucleotide sequence of the antisense strand. The phrase "completely complementary" may apply to cases where the oligonucleotide of the longer sense strand and the antisense strand includes a nucleotide sequence that is completely complementary to the oligonucleotide of the shorter strand.

[0028] In an embodiment, the double-stranded ribonucleic acid also includes a modified nucleotide as described below (see also Table 1). Therefore, the term "nucleotide" as used herein is intended to refer not only to guanosine 3'-phosphate, cytidine 3'-phosphate, adenosine 3'-phosphate, and uridine 3'-phosphate, but also encompasses various modified nucleotides.

[0029] The term "double-stranded ribonucleic acid" or "dsRNA" as used herein refers to a ribonucleic acid (RNA) molecule having a double-stranded structure comprising two antiparallel, substantially complementary oligonucleotides or a complex thereof. Examples of double-stranded ribonucleic acids as used herein include, but are not limited to, siRNA (small interfering RNA). In an embodiment, the double-stranded ribonucleic acid comprises a sense strand and an antisense strand. By RNAi using the double-stranded ribonucleic acid in an embodiment, the mRNA of the C5 component gene is cleaved as a target mRNA molecule in the RISC complex, and as a result, the expression of the C5 component is suppressed. For example, the expression of the C5 component in cells of a subject is suppressed.

[0030] The double-stranded ribonucleic acid of the embodiment can be synthesized, for example, using a chemical synthesis method known in the art (e.g., described in Nucleic Acid Research, 35(10), 3287-96 (2007)), and enzymatic transcription.

[0031] The double-stranded ribonucleic acid of the embodiment includes various modifications. The modification can be performed using a method known in the art. Examples of the modification include modification of sugar.

[0032] Examples of sugar modification include modification of the ribose moiety constituting the ribonucleoside, in particular substitution or addition of a hydroxy group at the 2'-position, more particularly a 2'-O-methyl-modified nucleotide in which the hydroxy group has been substituted with a methoxy group. The nucleotides represented by the lowercase letters a, u, g and c in Table 1 are 2'-O-methyl-modified nucleotides, and each of the sense strand and the antisense strand of the double-stranded ribonucleic acid of the embodiment comprises a 2'-O-methyl-modified nucleotide.

[0033] Double-stranded ribonucleic acid can be modified by inserting an additional nucleotide or nucleotide derivative, called an overhang, on the 3' side or 5' side of the region where the sense strand and antisense strand form a double strand. The double-stranded ribonucleic acid of an embodiment comprises a sense strand and/or an antisense strand comprising deoxythymidine (dT) at the 3' end of SEQ ID NO: 13 and SEQ ID NO: 14, and a sense strand and an antisense strand comprising inverted deoxythymidine (idT), as in SEQ ID NO: 129. The double-stranded ribonucleic acid of an embodiment also comprises a sense strand and/or an antisense strand comprising U, A, etc., added as an overhang sequence, for example, which comprises UUUU added at the 3' end of the antisense strand, as in SEQ ID NO: 140 and 142.

[0034] Alternatively, the double-stranded ribonucleic acid may have a modified backbone by modifying or substituting a phosphodiester bond. Examples of modifying or substituting a phosphodiester bond include a phosphorothioate bond. The double-stranded ribonucleic acid of an embodiment also includes a double-stranded ribonucleic acid comprising adjacent nucleotides linked by a phosphorothioate bond, as in SEQ ID NO: 13; SEQ ID NO: 14, and SEQ ID NO: 121.

[0035] (Lipid complex)

A pharmaceutical composition according to an embodiment of the present invention comprises a lipid complex comprising a double-stranded ribonucleic acid. In an embodiment, the lipid complex comprises (I) the above-mentioned double-stranded ribonucleic acid, (II) a cationic lipid, and (III) at least one lipid selected from the group consisting of a neutral lipid, a polyethyleneglycol-modified lipid (PEG lipid), and a sterol. Examples of the lipid complex herein include, but are not limited to, LNPs (lipid nanoparticles). In a particular embodiment, the pharmaceutical composition comprises a lipid complex encapsulating a double-stranded ribonucleic acid. The pharmaceutical composition according to another embodiment comprises a lipid nanoparticle comprising a double-stranded ribonucleic acid.

[0036] Examples of the form of the lipid complex formed by a lipid containing a cationic lipid and a double-stranded ribonucleic acid include a complex of a double-stranded ribonucleic acid and a membrane consisting of a lipid monolayer (one type of molecule) (reverse micelle); a complex of a double-stranded ribonucleic acid and a liposome; and a complex of a double-stranded ribonucleic acid and a micelle. In the lipid complex according to an embodiment of the present invention, the double-stranded ribonucleic acid is encapsulated in a fine particle containing a lipid containing a cationic lipid.

[0037] In an embodiment, the lipid complex comprises double-stranded ribonucleic acid in an amount of, for example, from 0.01 to 50% by weight, from 0.1 to 30% by weight, or from 1 to 10% by weight of the total weight of the lipid complex.

[0038] The cationic lipid is an amphiphilic molecule with a lipophilic region comprising one or more hydrocarbon groups and a hydrophilic region comprising a polar group that undergoes protonation at a certain pH. Examples of the cationic lipid of an embodiment include, but are not specifically limited to, the cationic lipids described in International Application Publication Nos. WO 2015/105131, WO 2016/104580, and WO 2017/222016, and alternatively, a cationic lipid with improved biodegradability described in International Application Publication No. WO 2016/104580 or WO 2017/222016 can be used. Examples of the cationic lipid of the embodiment include 1-oxo-1-(undecan-5-yloxy)nonadecan-10-yl-1-methylpiperidine-4-carboxylate, 1-((2-butyloctyl)oxy)-1-oxononadecane-10-yl-1-methylpiperidine-4-carboxylate, 1-oxo-1-(undecan-5-yloxy)heptadecan-8-yl-1-methylpiperidine 4-carboxylate, 21-oxo-21-(undecan-5-yloxy)heneicosan-10-yl-1-methylpiperidine-4-carboxylate, 21-(octan-3-yloxy)-21-oxogeneicosan-10-yl-1-methylpiperidine-4-carboxylate, 1-((2-Butyloctyl)oxy)-1-oxoicosan-10-yl-1-methylpiperidine-4-carboxylate, (Z)-1-((2-Butylnon-3-en-1-yl)oxy)-1-oxoicosan-10-yl-1-methylpiperidine-4-carboxylate, 1-oxo-1-((3-pentyloctyl)oxy)icosan-10-yl-1-methylpiperidine-4-carboxylate, 1-((3,4-dipropylheptyl)oxy)-1-oxocosan-10-yl-1-methylpiperidine-4-carboxylate, 1-((6-(butyldisulfanyl)-3-(3-(butyldisulfanyl)propyl)hexyl)oxy)-1-oxoicosan-10-yl-1-methylpiperidine-4-carboxylate, 2-butyloctyl-10-((4-(dimethylamino)butanoyl)oxy)icosanoate, 2-{9-[(2-butyloctyl)oxy]-9-oxononyl}dodecyl 1-methylpiperidine-4-carboxylate, 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate, 2-nonyl-11-oxo-11-[(3-pentyloctyl)oxy]undecyl 1-methylpiperidine-4-carboxylate, bis(3-pentyloctyl)-9-{[(1-methylpiperidin-4-carbonyl)oxy]methyl}heptadecanedioate, di[(Z)-2-nonen-1-yl]-9-{[(1-methylpiperidin-4-carbonyl)oxy]methyl}heptadecanedioate, 1-(2-octylcyclopropyl)heptadecan-8-yl-1-methylpiperidine-4-carboxylate, (3S)-2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl-1-methylpyrrolidine-3-carboxylate and (3R)-2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl-1-methylpyrrolidine-3-carboxylate. In one embodiment, the cationic lipid is 1-((2-butyloctyl)oxy)-1-oxoicosan-10-yl-1-methylpiperidine-4-carboxylate, 1-((2-butyloctyl)oxy)-1-oxononadecane-10-yl-1-methylpiperidine-4-carboxylate, 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate, 1-(2-octylcyclopropyl)heptadecan-8-yl-1-methylpiperidine-4-carboxylate, (3S)-2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpyrrolidine-3-carboxylate, or (3R)-2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpyrrolidine-3-carboxylate. In a particular embodiment, the cationic lipid is 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate.

[0039] In an embodiment, the lipid complex comprises the above-described cationic lipid in an amount of, for example, 10 to 100 mol %, 20 to 90 mol %, 30 to 90 mol %, or 40 to 70 mol %, based on the total amount of lipids contained in the lipid complex. One cationic lipid may be used alone, or a mixture of two or more cationic lipids may be used.

[0040] As a lipid component, the lipid complex according to an embodiment comprises: (I) the above-described cationic lipid and (II) at least one lipid selected from the group consisting of a neutral lipid, a polyethyleneglycol-modified lipid and a sterol. The lipid complex according to an embodiment comprises the lipid component in an amount of, for example, from 50 to 99.99% by weight, from 70 to 99.9% by weight or from 90 to 99% by weight of the total weight of the lipid complex.

[0041] The term "neutral lipid" refers to a lipid that is present at physiological pH either in an uncharged form or as a neutral zwitterion. Examples of a neutral lipid according to an embodiment of the present invention include a phospholipid and a ceramide. Examples of the phospholipid according to an embodiment of the present invention include DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), HSPC (hydrogenated soybean phosphatidylcholine), SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), EPC (egg phosphatidylcholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DAPC (1,2-diarachidoyl-sn-glycero-3-phosphocholine), DBPC (1,2-dibehenoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPG (1,2-dioleoyl-sn-glycero-3-phosphoglycerol), DPPG (1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol), DSPG (1,2-distearoyl-sn-glycero-3-phosphoglycerol), DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), DOPE-MAL (N-(3-maleimide-1-oxopropyl)-L-α-phosphatidylethanolamine, dioleoyl) and sphingomyelin. In an embodiment, the neutral lipid is DOPE, HSPC, DPPC, DSPC or DAPC. In a particular embodiment, the neutral lipid is DSPC. One neutral lipid may be used alone, or a mixture of two or more neutral lipids may be used.

[0042] In an embodiment, the lipid complex may comprise a neutral lipid in an amount of, for example, 0 to 50 mol %, 0 to 40 mol %, 0 to 30 mol %, or 0 to 20 mol %, based on the total amount of lipids contained in the lipid complex. In another embodiment, the lipid complex may comprise a neutral lipid in an amount of, for example, 0.1 to 20 mol %, or 3 to 15 mol %, based on the total amount of lipids contained in the lipid complex.

[0043] A polyethylene glycol-modified lipid (PEG lipid) refers to a lipid having a polyethylene glycol group. In an embodiment, examples of the polyethylene glycol-modified lipid (PEG lipid) include PEG2000-DMG, PEG2000-DPG, PEG2000-DSG, PEG5000-DMG, PEG5000-DPG, PEG5000-DSG, PEG-cDMA, PEG-C-DOMG, PEG-DAG, PEG-DAA, PEG-phospholipid, PEG-cholesterol, and PEG-ceramide (Cer). Examples of PEG2000-DMG, PEG2000-DPG, PEG2000-DSG, PEG-cDMA, and PEG-C-DOMG. In a particular embodiment, the polyethylene glycol-modified lipid is PEG2000-DMG. PEG in the present description also includes methoxy-PEG (MPEG). Specifically, for example, MPEG2000-DMG is included in PEG2000-DMG, and MPEG2000-DPG is also included in PEG2000-DPG. One polyethyleneglycol-modified lipid may be used alone, and a mixture of two or more polyethyleneglycol-modified lipids may also be used.

[0044] In an embodiment, the lipid complex can comprise a polyethyleneglycol-modified lipid in an amount of, for example, 0 to 30 mol %, 0 to 20 mol %, 0 to 10 mol %, or 0.5 to 2 mol %, based on the total amount of lipids contained in the lipid complex. In another embodiment, the lipid complex can comprise a polyethyleneglycol-modified lipid in an amount of, for example, 0.01 to 10 mol %, or 0.1 to 3 mol %, based on the total amount of lipids contained in the lipid complex.

[0045] The sterol is an alcohol having a steroid backbone. Examples of a sterol in an embodiment include cholesterol, dihydrocholesterol, lanosterol, β-sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, fucosterol, and 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol). In one embodiment, the sterol is cholesterol, dihydrocholesterol, lanosterol, or β-sitosterol. In a particular embodiment, the sterol is cholesterol. One sterol may be used alone, or a mixture of two or more sterols may be used.

[0046] In an embodiment, the lipid complex may comprise sterol in an amount of, for example, 0 to 90 mol %, 10 to 80 mol %, or 20 to 40 mol %, based on the total amount of lipids contained in the lipid complex. In another embodiment, the lipid complex may comprise sterol in an amount of, for example, 0.1 to 70 mol %, or 15 to 60 mol %, based on the total amount of lipids contained in the lipid complex.

[0047] In an embodiment, the combination of lipid components in the lipid complex is not particularly limited, and examples thereof include a combination of the above-described cationic lipid, neutral lipid and sterol, and a combination of the above-described cationic lipid, neutral lipid, polyethylene glycol-modified lipid and sterol.

[0048] In one embodiment, the lipid complex comprises a cationic lipid, a neutral lipid, a polyethyleneglycol-modified lipid, and a sterol. The cationic lipid is necessary for encapsulating the nucleic acid or efficiently delivering the nucleic acid to target cells, and it has been reported that the polyethyleneglycol-modified lipid is necessary to prevent particle aggregation (Molecular Therapy-Nucleic Acids (2012)1, e37). Moreover, it has been reported that the coexistence of four lipids: a neutral lipid and a sterol in addition to the two lipids mentioned above is critical for encapsulating the nucleic acid to form stable particles (Nanoscale. 2019 Nov 21; 11(45): 21733-21739.).

[0049] In an embodiment, the lipid complex can be a lipid complex comprising, for example, 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl-1-methylpiperidine-4-carboxylate and at least one lipid selected from the group consisting of DSPC, PEG2000-DMG, and cholesterol, or a lipid complex comprising 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl-1-methylpiperidine-4-carboxylate, DSPC, PEG2000-DMG, and cholesterol.

[0050] In an embodiment, the lipid complex encapsulating the double-stranded ribonucleic acid comprises lipid components that are cationic lipid/neutral lipid/polyethyleneglycol-modified lipid/sterol, and wherein the molar ratio of the lipids can be, for example, 10-99/0-50/0-10/0-50, 10-99/1-50/0.5-10/10-50, 40-70/1-20/0.5-2/20-40, or 40-70/0-20/0.5-2/20-40. In an embodiment, the molar ratio of cationic lipid/neutral lipid/polyethyleneglycol-modified lipid/sterol in the lipid complex is 30-90/0.1-20/0.01-10/0.1-70. In another embodiment, the molar ratio of cationic lipid/neutral lipid/polyethyleneglycol-modified lipid/sterol in the lipid complex is 40-70/3-15/0.1-3/15-60. In a particular embodiment, the molar ratio of cationic lipid/neutral lipid/polyethyleneglycol-modified lipid/sterol in the lipid complex is 60/10.5/1.5/28.

[0051] In the present description, the "average particle size" of the lipid complex refers to the Z-average particle size, and the average particle size is measured by a dynamic light scattering method. The average particle size (Z-average) of the lipid complex containing double-stranded ribonucleic acid may be, for example, from 10 to 1000 nm, from 30 to 500 nm, or from 30 to 200 nm, although the average particle size is not particularly limited to these values. In an embodiment, the average particle size of the lipid complex containing double-stranded ribonucleic acid is 100 nm or less. In a specific embodiment, the average particle size of the lipid complex containing double-stranded ribonucleic acid is 65 nm or greater and 100 nm or less. In another embodiment, the average particle size of the lipid complex containing double-stranded ribonucleic acid is 80 nm or greater and 100 nm or less, and in yet another embodiment, the average particle size of the lipid complex containing double-stranded ribonucleic acid is 85 nm or greater and 100 nm or less.

[0052] In an embodiment, the pH of the lipid complex solution is 5.0 or less or 7.5 or greater. In another embodiment, the pH of the lipid complex solution is 2.0 or greater and 5.0 or less or 7.5 or greater and 11.0 or less. In another embodiment, the pH of the lipid complex solution is 2.0 or greater and 5.0 or less, 2.5 or greater and 5.0 or less, 3.0 or greater and 5.0 or less, 3.5 or greater and 5.0 or less, 4.0 or greater and 5.0 or less, 4.5 or greater and 5.0 or less, 7.5 or greater and 11.0 or less, 7.5 or greater and 10.5 or less, 7.5 or greater and 10.0 or less, 7.5 or greater and 9.5 or less, 7.5 or greater and 9.0 or less, or 7.5 or greater and 8.5 or less. In a particular embodiment, the pH of the lipid complex solution is 7.5 or greater and 8.5 or less. The storage stability of the pharmaceutical composition is improved by setting the pH of the lipid complex solution in the above range.

[0053] The storage stability of a pharmaceutical composition according to an embodiment of the present invention can be determined, for example, based on the degree of change in average particle size by comparing the average particle size of the lipid complex before storage and the average particle size of the lipid complex after storage for a given period of time. As used herein, "before storage" may mean, for example, the time immediately after the pharmaceutical composition is prepared or, if the pH has been adjusted, the time immediately after the pH has been adjusted. Examples of storage conditions for a pharmaceutical composition according to an embodiment of the present invention include conditions allowing it to be stored in a cool place or a refrigerator (refrigerated storage conditions), as well as normal temperature or room temperature. In an embodiment, the storage conditions of the pharmaceutical composition are from 2 to 8°C. In another embodiment, the storage conditions of the pharmaceutical composition are 5°C. In yet another embodiment, the storage conditions of the pharmaceutical composition are 25°C.

[0054] The storage stability of a pharmaceutical composition can be determined, for example, based on the average particle size of the lipid complex 2 weeks after the start of storage (after 2 weeks of storage), or determined based on the average particle size of the lipid complex 1 month after the start of storage (after 1 month of storage), or determined based on the average particle size of the lipid complex 2 months after the start of storage (after 2 months of storage), or determined based on the average particle size of the lipid complex 3 months after the start of storage (after 3 months of storage). In an embodiment, the storage stability can be considered good if the change in the average particle size of the lipid complex after storage is ±10% or less than the average particle size of the lipid complex before storage, the storage stability can be considered good if the change in the average particle size of the lipid complex after storage is ±8% or less than the average particle size of the lipid complex before storage, and the storage stability can be considered good if the change in the average particle size of the lipid complex after storage is ±5% or less than the average particle size of the lipid complex before storage. In particular, for example, the storage stability of a pharmaceutical composition can be considered good if the average particle size of the lipid complex after 2 weeks from the start of storage differs by ±10% or less from the average particle size of the lipid complex before storage. In another embodiment, the storage stability of the pharmaceutical composition can be determined based on the increase in the average particle size, and, for example, the storage stability can be considered good if the average particle size of the lipid complex after storage for a given period of time differs by +10% or less from the average particle size of the lipid complex before storage, the storage stability can be considered good if the average particle size of the lipid complex after storage for a given period of time differs by +8% or less from the average particle size of the lipid complex before storage, and the storage stability can be considered good if the average particle size of the lipid complex after storage for a given period of time differs by +5% or less from the average particle size of the lipid complex before storage. In particular, for example, the storage stability can be considered good if the average particle size of the lipid complex after 2 weeks from the start of storage differs by +10% or less from the average particle size of the lipid complex before storage.

[0055] In an embodiment, the encapsulation efficiency of siRNA for a lipid complex encapsulating double-stranded ribonucleic acid can be calculated, for example, based on the concentration of siRNA in the formulation diluted with RNase-free water, which is taken as the concentration of siRNA in the outer LNP solution, and based on the concentration of siRNA in the formulation diluted with 1% Triton X-100, which is taken as the total concentration of siRNA in the formulation, where each siRNA concentration is measured using the Quant-iT RiboGreen RNA reagent (Invitrogen, Cat. No. R11491) (see also Kewal K. Jain, Drug Delivery System, Methods in Molecular Biology, Vol. 1141: 109-120). The encapsulation efficiency calculated in this manner is, for example, greater than 80%, greater than 85%, or greater than 90%. In an embodiment, the encapsulation efficiency of siRNA for the lipid complex of the embodiment is greater than 90%.

[0056] <Method for obtaining a lipid complex>

Examples of methods for encapsulating an effective molecule in a lipid complex include a reverse-phase evaporation method, a zwitterion (NaCl) hydration method, a cationic core hydration method, and a method using ethanol and calcium (see Biomembr., 1468, 239-252 (2000)). The lipid complex encapsulating the double-stranded ribonucleic acid in the pharmaceutical composition of the embodiment can be prepared using any of these methods known in the art.

[0057] In an embodiment, a lipid complex comprising a double-stranded ribonucleic acid can be obtained, for example, by mixing a lipid solution comprising a cationic lipid and at least one lipid selected from the group consisting of a neutral lipid, a polyethylene glycol-modified lipid, and a sterol, and an acidic buffer comprising a double-stranded ribonucleic acid. Using such a method, a lipid complex can be obtained, the interior of which is filled with a core of double-stranded ribonucleic acid and lipids. The lipid complex according to an embodiment comprising a double-stranded ribonucleic acid can comprise a cationic lipid and at least one lipid selected from the group consisting of a neutral lipid, a polyethylene glycol-modified lipid, and a sterol.

[0058] A lipid complex according to an embodiment comprising a double-stranded ribonucleic acid can be obtained using a method comprising: a step of (a) mixing a polar organic solvent-containing aqueous solution comprising (I) a cationic lipid and (II) at least one lipid selected from the group consisting of a neutral lipid, a polyethyleneglycol-modified lipid, and a sterol, and an aqueous solution comprising (III) a double-stranded ribonucleic acid, to obtain a mixed solution; and a step of (b) reducing the content of the polar organic solvent in the mixed solution.

[0059] A lipid complex encapsulating the double-stranded ribonucleic acid in a fine particle containing lipids can be formed by electrostatic interaction between the double-stranded ribonucleic acid and the cationic lipid, each of which is water-soluble, and hydrophobic interaction between the lipids. For example, the lipid complex can be formed by decreasing the content of a polar organic solvent in a mixed solution to change the solubility of a lipid component containing (I) a cationic lipid and (II) at least one lipid selected from the group consisting of a neutral lipid, a polyethylene glycol-modified lipid, and a sterol in an aqueous solution containing a polar organic solvent. Examples of the polar organic solvent include an alcohol such as ethanol.

[0060] First, in step (a), a polar organic solvent-containing aqueous solution containing (I) a cationic lipid and (II) at least one lipid selected from the group consisting of a neutral lipid, a polyethylene glycol-modified lipid and a sterol dissolved therein is mixed with an aqueous solution containing (III) a double-stranded ribonucleic acid to obtain a mixed solution. The concentration of the polar organic solvent in the polar organic solvent-containing aqueous solution is not particularly limited as long as the conditions for dissolving the lipid molecules are satisfied even after mixing with the aqueous solution containing the double-stranded ribonucleic acid. The concentration of the polar organic solvent in the mixed solution obtained in step (a) may be from 0.1 to 60% by weight. An aqueous solution containing (III) double-stranded ribonucleic acid is obtained, for example, by dissolving double-stranded ribonucleic acid in an acidic buffer.

[0061] Subsequently, in step (b), the content of the polar organic solvent is reduced by adding water or the like to the mixed solution. Due to this, a lipid complex can be formed. In order to efficiently form the lipid complex, it is preferable to rapidly reduce the content of the polar organic solvent. As an example, the concentration of the polar organic solvent in the final aqueous solution containing the polar organic solvent in step (b) can be 0 to 5% by weight.

[0062] The mixed solution obtained in step (a) can be dialyzed to remove the polar organic solvent and replace the solvent with a pharmaceutically acceptable medium. The content of the polar organic solvent in the solution decreases during dialysis, as a result of which a lipid complex can be formed.

[0063] By using the method for producing the composition according to the embodiment, it is possible to obtain a lipid complex encapsulating double-stranded ribonucleic acid in the interior of a fine particle with high encapsulation efficiency.

[0064] Examples of the acidic buffer for dissolving the double-stranded ribonucleic acid therein include a sulfate buffer, a phosphate buffer, a phthalate buffer, a tartrate buffer, a citrate buffer, a formate buffer, an oxalate buffer, and an acetate buffer.

[0065] Examples of the solvent for dissolving lipids therein include a polar organic solvent such as an alcohol, and the solvent may be, for example, ethanol, isopropanol, chloroform or tert-butanol.

[0066] The method according to an embodiment of the present invention for producing a pharmaceutical composition may further comprise adjusting the pH of the lipid complex solution. Adjusting the pH of the solution is typically performed after incorporating the double-stranded ribonucleic acid into the lipid complex. The adjusting step may be, for example, adjusting the pH of the lipid complex solution to 5.0 or less or 7.5 or more, or adjusting the pH of the lipid complex solution to 2.0 or more and 5.0 or less or 7.5 or more and 11.0 or less, or adjusting the pH of the lipid complex solution to 2.0 or more and 5.0 or less, or adjusting the pH of the lipid complex solution to 7.5 or more and 11.0 or less, or adjusting the pH of the lipid complex solution to 7.5 or more or 10.0, or adjusting the pH of the lipid complex solution to 7.5 or more and 9.5 or less, or adjusting the pH of the lipid complex solution to 7.5 or more and 9.0 or less, or adjusting the pH of the lipid complex solution to 7.5 or more and 8.5 or less. In a specific embodiment, the adjusting step comprises adjusting the pH of the lipid complex solution to 7.5 or greater and 8.5 or less.

[0067] The pH adjustment can be carried out using a known method and can be carried out, for example, using an acidic aqueous solution of hydrochloric acid, etc., or an alkaline aqueous solution of sodium hydroxide, etc., or carried out using a buffer solution (buffer) of phosphate, citrate, acetate, tartrate, borate, etc.

[0068] As described above, the expression of complement component C5 can be inhibited by RNAi using double-stranded ribonucleic acid contained in a pharmaceutical composition according to an embodiment of the present invention. In an embodiment, the pharmaceutical composition can comprise a pharmaceutically acceptable carrier in addition to a lipid complex containing double-stranded ribonucleic acid.

[0069] Examples of the pharmaceutically acceptable carrier include liquid or solid fillers, diluent, excipients, formulation aids, and solvent encapsulating materials.

[0070] In an embodiment, the pharmaceutical composition may be, for example, in the form of a powder obtained by removing the solvent by freeze-drying, etc., or in the form of a liquid. The pharmaceutical composition of an embodiment is a powder composition comprising a lipid complex of any of the above-described embodiments. The powder composition can be obtained by removing the solvent from the composition in the form of a liquid (dispersion), for example, by filtration or centrifugation, or obtained by freeze-drying the dispersion. In case the pharmaceutical composition is in the form of a powder, the pharmaceutical composition can be suspended or dissolved in a pharmaceutically acceptable medium before use and used as an injection. The pharmaceutical composition of an embodiment is a liquid composition comprising a lipid complex of any of the above-described embodiments and a pharmaceutically acceptable medium. If the pharmaceutical composition is presented in the form of a liquid, the pharmaceutical composition can be directly used as an injection preparation or suspended or dissolved in a pharmaceutically acceptable medium and used as an injection preparation.

[0071] When administered to a subject in need thereof, a pharmaceutical composition of an embodiment of the present invention can inhibit expression of complement component C5 in the subject via RNAi. As used herein, a "subject in need thereof" refers to a subject having a disease or disorder associated with expression or activity of the gene for component C5, or a subject who is determined to be at high risk for developing the same.

[0072] In some embodiments, the double-stranded ribonucleic acid that is contained in the pharmaceutical composition of an embodiment of the present invention can suppress the expression of complement component C5, and therefore, the pharmaceutical composition of an embodiment of the present invention can be useful for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). Thus, in other embodiments, the present invention encompasses a method of treating paroxysmal nocturnal hemoglobinuria or atypical hemolytic uremic syndrome, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of the embodiment.

[0073] The subject to which the pharmaceutical composition of the embodiment is administered is not limited, and, for example, the present invention can be applied to humans or non-human mammals (such as monkeys, mice, rats, rabbits, cows, horses, goats).

[0074] The method of administering the pharmaceutical composition of the embodiment to a subject (e.g., route of administration, dose, frequency of administration per day, time of administration) is not limited and can be appropriately determined by a person of ordinary skill in the art (e.g., doctors) in accordance with the health condition of the subject, the stage of the disease, the type of agent to be used in combination.

[0075] (Route of administration)

The method of administration of the pharmaceutical composition according to the embodiment is not particularly limited and may be parenteral administration, examples of which include intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration and intrathecal administration.

[0076] The pharmaceutical composition of the embodiment can be administered in an amount sufficient to inhibit complement component C5 depending on the route of administration. The dose of the pharmaceutical composition of the embodiment can be, for example, from 0.01 mg to 100 mg, or from 0.1 mg to 50 mg, or from 0.3 mg to 10 mg per kg of body weight of the subject.

[0077] It will be understood by a person of ordinary skill in the art that the present invention may be implemented by an appropriate combination of any one or more of all embodiments described herein, except where the combination causes any technical contradiction. In addition, it will be understood by a person of ordinary skill in the art that it would be preferable to implement the present invention by an appropriate combination of any one or more of all preferred or advantageous embodiments described herein, except where the combination causes any technical contradiction.

[0078] The entire content disclosed in the literature references cited herein is incorporated by reference in its entirety, and a person of ordinary skill in the art can cite and understand the corresponding content disclosed in the literature as part of the present description in accordance with the context of the present description without departing from the spirit and scope of the present invention.

[0079] The literature cited herein is provided solely for the purpose of disclosing prior art prior to the filing date of this application and should not be interpreted as an admission that the present inventors are not entitled to priority to the disclosures due to any earlier invention or for any other reason. All descriptions in the literature are based on information that was available to the applicant and in no way constitute an admission that the content described is correct.

[0080] The terms used herein are intended to describe particular embodiments and are not intended to limit the present invention.

[0081] As used herein, the term "comprise" is intended to indicate the presence of the referenced entity (e.g., member, step, element, or number) unless the context clearly requires otherwise, and does not exclude the presence of another entity (e.g., member, step, element, or number). The term "consist of" encompasses embodiments described by the term(s) "consist of" and/or "consist essentially of."

[0082] Unless otherwise defined, all terms (including technical terms and scientific terms) used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention pertains. Each term used herein should be interpreted as having a meaning consistent with the meaning in the present description and in related art fields, unless otherwise specified, and should not be interpreted in terms of an idealized or overly literal meaning.

[0083] Although terms such as "first" and "second" are used to represent various elements, it should be understood that such elements should not be limited by these terms per se. These terms are used only to distinguish one element from another element, and, for example, it is within the scope of the present invention to refer to a first element as a "second element" and similarly to refer to a second element as a "first element."

[0084] It should be understood that the numerical values used herein to indicate component contents, numerical ranges, etc., are modified by the term "about" unless otherwise indicated. For example, "4°C" is understood to mean "about 4°C" unless otherwise indicated, and of course, one of ordinary skill in the art can reasonably understand the allowable deviation in accordance with the general knowledge and context of the present description. As used herein, "about" is intended to mean applying conventional rounding techniques to take into account significant digits of each numerical value. In particular, "about" is used to further include numerical values rounded to the nearest whole number, where, for example, the range corresponding to "about 4" is from 3.5 to 4.4, and is used to further include numerical values rounded to one decimal place, where, for example, the range corresponding to "about 4.5" is from 4.45 to 4.54. Cases of three or more digits after the decimal point are understood in a similar way.

[0085] Unless the context clearly indicates otherwise, the singular forms of embodiments used herein and in the claims shall be understood as admitting the plural form and vice versa, provided that this does not cause a technical contradiction.

[0086] Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention can be implemented in various embodiments and should not be interpreted as being limited to the examples described below. A person of ordinary skill in the art can implement the present invention with various modifications, additions, deletions, substitutions, etc. without changing the spirit or scope of the present invention.

[Examples]

[0087] [Example 1. In vitro screening in case of single administration (1)]

(Obtaining double-stranded nucleic acids)

The sense strands and antisense strands listed in Table 2 were synthesized using the phosphoramidite method and then hybridized with double-stranded nucleic acid synthesis (GeneDesign, Inc.). Sequence abbreviations are as shown in Table 1. Each double-stranded nucleic acid synthesized had a hydroxy group at each 3' end instead of a phosphate group.

[0088]

[Table 1]

Reduction Nucleotide A Adenosine 3'-phosphate U Uridine 3'-phosphate G Guanosine 3'-phosphate C Cytidine 3'-phosphate a 2'-O-methyladenosine-3'-phosphate u 2'-O-methyluridine-3'-phosphate g 2'-O-methylguanosine-3'-phosphate c 2'-O-methylcytidine-3'-phosphate dT Deoxythymidine (idT) Inverted deoxythymidine (inverted dT) ^ Phosphothioate bond

The absence of a symbol indicates that the nucleotides are linked to each other via a phosphodiester bond.

[0089]

[0090] (In vitro screening)

Each of the double-stranded nucleic acids listed in Table 2, in combination with Lipofectamine RNAiMax transfection reagent (Invitrogen, catalog number: 13778150), was diluted with Opti-MEM medium (Gibco, catalog number: 31985062) to obtain a mixed siRNA/RNAiMax solution with a final concentration of 3 nM double-stranded nucleic acid and 0.3% RNAiMax. The siRNA/RNAiMax mixed solution was added in 20 μl aliquots into the wells of a 96-well culture plate, and Hep3B cells (obtained from ATCC), a human liver cancer-derived cell line, were seeded into each well at 20,000 cells/80 μl/well and cultured under conditions of 37°C and 5% _CO2 overnight. The cultured cells were prepared into a lysate containing a template for real-time PCR using CellAmp (registered trademark) RT-PCR Direct RNA Preparation Kit (from Takara Bio Inc., Cat. No.: 3732) and Proteinase K (from Takara Bio Inc., Cat. No.: 9034) according to the protocol provided by Takara Bio Inc. Thereafter, cDNA was prepared using PrimeScript (registered trademark) RT Master Mix (Perfect Real Time) (from Takara Bio Inc., catalog no.: RR036A) according to the protocol provided by Takara Bio Inc. In addition, Ct values were measured for the target gene of human C5 component and the endogenous control gene of human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) using EagleTaq Universal Master Mix (ROX) (from Roche Diagnostics KK, catalog no.: 07260296190) and TaqMan probe (from Applied Biosystems, C5: Hs00156197_m1; GAPDH: Hs02758991_g1) with ABI7900HT Real-Time PCR System (from Applied Biosystems) according to the protocol provided by Applied Biosystems. The expression level of C5 mRNA in the case where Hep3B cells were treated with transfection reagent alone without adding siRNA was defined as 100%, and the residual C5 mRNA level (relative value) was calculated for each siRNA injection using the calibration curve method. A mock control that does not overlap with any human gene was used as a negative control.

[0091] The results are shown in Table 3.

[0092]

[0093] [Example 2. In vitro screening in case of single administration (2)]

(Obtaining double-stranded nucleic acids)

The sense strands and antisense strands listed in Table 4 were synthesized using the phosphoramidite method and then hybridized with double-stranded nucleic acid synthesis (GeneDesign, Inc.).

[0094]

[0095] (In vitro screening)

A test was performed to measure the Ct values of the target gene of human C5 component and the endogenous control gene of human GAPDH in cultured Hep3B cells in the same manner as in Example 1, except that the siRNA/RNAiMax mixed solution was prepared at a final concentration of 1 nM double-stranded nucleic acid and 0.3% RNAiMax. As in Example 1, the expression level of C5 component mRNA in the case of using only the lipofection reagent was determined as 100%, and the residual level of C5 component mRNA (relative value) was calculated for each siRNA injection.

[0096] The results are shown in Table 5. For all double-stranded nucleic acids except siRNA-008-29, reduced residual levels of C5 component mRNA were detected, indicating downregulation of C5 component expression.

[0097]

[0098] [Example 3. In vivo screening (sequence search)]

(Obtaining siRNA-LNP)

Each siRNA listed in Table 6 was dissolved in 10 mM sodium citrate (pH 4.0) to prepare a diluted siRNA solution. The lipid solution was prepared by dissolving 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate, DSPC (NIPPON FINE CHEMICAL CO., LTD.), cholesterol (NIPPON FINE CHEMICAL CO., LTD.), and MPEG2000-DMG (NOF CORPORATION) in ethanol at a molar ratio of 60/10.5/28/1.5. Lipid nanoparticles (LNPs) were prepared by mixing the diluted siRNA solution and lipid solution at a flow rate of 3 ml/min and 1 ml/min, respectively, with an siRNA/lipid weight ratio of 0.1. The outer solution, which was an aqueous solution of the obtained LNPs, was replaced with PBS (pH 7.4) by dialysis using a Float-A-Lyzer G2 (SPECTRUM, molecular weight cutoff 100 kDa). After dialysis, the resulting product was concentrated and filter sterilized for experimental use. The siRNA concentration and encapsulation efficiency were measured using Quant-iT RiboGreen RNA Detection Reagent (Invitrogen, Cat. No. R11491). To calculate the encapsulation efficiency, the siRNA concentration measured after dilution with RNase-free water was taken as the siRNA concentration present in the outer LNP solution, and the siRNA concentration measured after dilution with 1% Triton X-100 was taken as the total siRNA concentration in the formulation. The average particle size (Z-mean) was measured using a particle size analyzer (Malvern Panalytical Ltd., Zetasizer Nano ZS). The product quality assessment results for the resulting LNPs are shown in Table 7.

[0099]

[0101] (In Vivo Screening)

LNPs encapsulating PBS or siRNA listed in Table 6 were intravenously administered to BALB/c mice (6-week-old males, n=3 per group) via the tail vein at a dose of 0.1 mg/kg siRNA, and blood and liver tissue samples were collected under anesthesia at 5 days and 14 days after administration. From the liver tissue frozen in liquid nitrogen, total RNA was purified using the RNeasy Plus Mini Kit (Qiagen, Cat# 74106) according to the protocol provided by the manufacturer. Then, cDNA was prepared using PrimeScript RT Master Mix (Perfect Real Time) (Takara Bio Inc., Cat# RR036A) according to the protocol provided by the manufacturer. In addition, the Ct values of the target gene of mouse C5 component and the endogenous control gene of mouse GAPDH were measured using TaqMan Gene Expression Master Mix (registered trademark) (Applied Biosystem, Cat. No. 4369510) and TaqMan Probe (Applied Biosystems, C5: Mm01336776_g1; GAPDH: Mm99999915_g1) with the ABI7500 Fast System (Applied Biosystems) according to the protocol provided by the manufacturer. The residual mRNA level of C5 component in the liver 5 days after administration in the PBS administration group was defined as 100%, and the residual mRNA level of C5 component in the liver (relative value) was calculated for each siRNA administration group using the Ct comparison method. The results are shown in Table 8.

[0102]

[0103] Blood samples collected on each sample collection day were centrifuged at 3000 rpm for 15 minutes, and then the heparinized plasma, which was the supernatant, was collected and stored at -80°C. Thereafter, the content of the mouse C5 component in the blood plasma was quantified by ELISA. Specifically, a mouse anti-C5 antibody, BB5.1 (Hycult Biotech, Cat. No. HM1073-FS), used as a capture antibody, was diluted with PBS(-) (Wako Pure Chemical Industries, Ltd., No. 045-29795) to a final concentration of 2 μg/mL and added to an assay plate (Nunc, Cat. No. 442404) and incubated at 4°C overnight. Thereafter, a blocking solution (PBS(-) (Wako Pure Chemical Industries, Ltd.) containing 1% BSA (R&D systems, Inc., Cat. No. DY995)) was added, and the resulting product was incubated at room temperature for 1 hour. The blocking solution was removed, and three washings were performed with a washing solution (PBS(-) (Wako Pure Chemical Industries, Ltd.) containing 0.02% Tween20). The washing solution was removed, and then a heparinized blood plasma sample diluted with the blocking solution was added, and the resulting product was incubated at room temperature for 5 hours. The blood plasma from the PBS administration group was used as a standard sample. The sample was removed and then washed five times with wash solution, and goat anti-human C5 antibody (Quidel Corporation, Cat. No. A306) diluted 4,000-fold in blocking solution was added and the resulting product was incubated at room temperature for 1 hour. The antibody was removed and then washed five times with wash solution, and HRP-labeled donkey anti-goat IgG (H+L) antibody (Jackson ImmunoResearch Inc., Cat. No. 805-035-180) diluted 40,000-fold in blocking solution was added and the resulting product was incubated at room temperature for 1 hour. The antibody was removed and then washed five times with wash solution. Thereafter, equal amounts of peroxidase substrate, TMB (3,3',5,5'-tetramethylbenzidine) (Kirkegaard & Perry Laboratories, Inc., Cat. No. 50-76-01), and peroxidase substrate solution B (Kirkegaard & Perry Laboratories, Inc., Cat. No. 50-65-00) were mixed together to prepare a detection _reagent , which was added and left to develop color. _H2SO4 (Wako Pure Chemical Industries, Ltd., Cat. No. 198-09595) was added as a stop solution, and then the absorbance at 450 nm and 650 nm was measured. Table 9 shows the relative values for the samples, with the concentration of the C5 component in the blood plasma determined as 100% 5 days after administration for the PBS administration group.

[0104] [Example 4. In vitro screening]

The sense strands and antisense strands listed in Table 10 were synthesized using the phosphoramidite method and then hybridized with double-stranded nucleic acid synthesis (GeneDesign, Inc.). The assay for measuring the Ct values of the human C5 target gene and the endogenous human GAPDH control gene in cultured Hep3B cells was carried out in the same manner as in Example 1, except that the siRNA/RNAiMax mixed solution was prepared at a final concentration of 0.003-10 nM double-stranded nucleic acid and 0.3% RNAiMax. As in Example 1, the expression level of the C5 component mRNA in the case of using only the lipofection reagent was determined as 100%, and the residual C5 component mRNA level (relative value) was calculated for each siRNA injection. The results are shown in Table 11.

[0105]

[0106]

[0107] [Example 5. In vivo screening (overhang modification)]

(Obtaining siRNA-LNP)

Lipid nanoparticles (LNPs) encapsulating siRNA were prepared in the same manner as in Example 3, except that the siRNAs listed in Table 12 were used. The product quality evaluation results for the obtained LNPs are presented in Table 13.

[0108]

[0109]

[0110] (In Vivo Screening)

LNPs encapsulating PBS or siRNA listed in Table 12 were intravenously administered to BALB/c mice (6-week-old males, n=3 per group) via the tail vein at a dose of 0.3 mg/kg siRNA, and blood and liver tissue samples were collected under anesthesia at 5 days, 14 days, and 21 days after administration. From the liver tissue frozen in liquid nitrogen, total RNA was purified using the RNeasy Plus Mini Kit (Qiagen, Cat# 74106) according to the protocol provided by the manufacturer. Then, cDNA was prepared using PrimeScript RT Master Mix (Perfect Real Time) (Takara Bio Inc., Cat# RR036A) according to the protocol provided by the manufacturer. In addition, the Ct values of the target gene of mouse C5 component and the endogenous control gene of mouse GAPDH were measured using TaqMan Gene Expression Master Mix (registered trademark) (Applied Biosystem, Cat. No. 4369510) and TaqMan Probe (Applied Biosystems, C5: Mm01336776_g1; GAPDH: Mm99999915_g1) with the ABI7500 Fast System (Applied Biosystems) according to the protocol provided by the manufacturer. The residual level of C5 component mRNA in the liver for each measurement day in the PBS administration group was defined as 100%, and the residual levels of C5 component mRNA in the liver (relative values) were calculated for each siRNA administration group using the Ct comparison method. The results are shown in Table 14.

[0111]

[0112] Blood samples collected on each sample collection day were centrifuged at 3000 rpm for 15 minutes, and then the heparinized plasma, which was the supernatant, was collected and stored at -80°C. Thereafter, the content of the mouse C5 component in the blood plasma was quantified by ELISA. Specifically, a mouse anti-C5 antibody, BB5.1 (Hycult Biotech, Cat. No. HM1073-FS), used as a capture antibody, was diluted with PBS(-) (Wako Pure Chemical Industries, Ltd., Cat. No. 045-29795) to a final concentration of 2 μg/mL and added to an assay plate (Nunc, Cat. No. 442404) and incubated at 4°C overnight. Thereafter, a blocking solution (PBS(-) (Wako Pure Chemical Industries, Ltd.) containing 1% BSA (R&D systems, Inc., Cat. No. DY995)) was added, and the resulting product was incubated at room temperature for 1 hour. The blocking solution was removed, and three washings were performed with a washing solution (PBS(-) (Wako Pure Chemical Industries, Ltd.) containing 0.02% Tween20). The washing solution was removed, and then a heparinized blood plasma sample diluted with the blocking solution was added, and the resulting product was incubated at room temperature for 5 hours. The blood plasma from the PBS administration group was used as a standard sample. The sample was removed and then washed five times with wash solution, goat anti-human C5 antibody (Quidel Corporation, Cat #A306) diluted 4,000-fold in blocking solution was added, and the resulting product was incubated at room temperature for 1 hour. The antibody was removed and then washed five times with wash solution, and HRP-labeled donkey anti-goat IgG (H+L) antibody (Jackson ImmunoResearch Inc., Cat #805-035-180) diluted 40,000-fold in blocking solution was added, and the resulting product was incubated at room temperature for 1 hour. The antibody was removed and then washed five times with wash solution. Thereafter, equal amounts of peroxidase substrate, TMB (3,3',5,5'-tetramethylbenzidine) (Kirkegaard & Perry Laboratories, Inc., Cat. No. 50-76-01), and peroxidase substrate solution B (Kirkegaard & Perry Laboratories, Inc., Cat. No. 50-65-00) were mixed together to prepare a detection _reagent , which was added and left to develop color. _H2SO4 (Wako Pure Chemical Industries, Ltd., Cat. No. 198-09595) was added as a stop solution, and then the absorbance at 450 nm and 650 nm was measured. Table 15 shows the relative values for the samples, with the concentration of the C5 component in the plasma determined as 100% on the day before administration for the PBS administration group.

[0113]

[0114] The residual mRNA levels of the C5 component in the liver and the concentrations of the C5 component in the blood plasma at 5 days, 14 days, and 21 days after administration were quantified and statistical analysis (t-test for independent groups) was performed for the siRNA-008-34 administration group and the siRNA-008 administration group. The results are shown in Fig. 1 and Fig. 2. The groups whose P-value is 0.05 or lower are marked with * (asterisk), and the groups whose P-value is 0.01 or lower are marked with **.

[0115] [Example 6. In vitro assay (sequence walk)]

The sense strands and antisense strands listed in Table 16 were synthesized using the phosphoramidite method and then hybridized with double-stranded nucleic acid synthesis (GeneDesign, Inc.). As in Example 1, the expression level of C5 mRNA using only the lipofection reagent was determined as 100%, and the residual C5 mRNA level (relative value) was calculated for each siRNA injection. The results are shown in Table 17.

[0116]

[0117]

[0118] [Example 7. Pharmacological test (hemolysis suppression effect)]

(Obtaining siRNA-LNP)

Lipid nanoparticles (LNPs) encapsulating siRNA were prepared in the same manner as in Example 3, except that the siRNAs listed in Table 18 were used. The product quality evaluation results for the obtained LNPs are presented in Table 19.

[0119]

[0120]

[0121] (In vivo assessment)

LNPs encapsulating PBS or siRNA listed in Table 18 were intravenously administered to BALB/c mice (6-week-old males, n=3 per group) via the tail vein at a dose of 1-3 mg/kg siRNA, and blood samples were collected under anesthesia at 5 days and 9 days after administration. Blood samples collected on each collection day were placed in a blood separation tube containing a clot activator (Immuno-Biological Laboratories Co, Ltd., Cat. No. 31203) and centrifuged at 3000 rpm for 15 minutes, and then the serum supernatant was collected and stored at -80°C. Thereafter, the complement activity in the serum was quantified as follows. Specifically, sheep red blood cells were prepared at a concentration of 1.5 × 10 ⁸ cells/mL using the CH50 Serum Complement Titer Test Kit (DENKA SEIKEN Co., Ltd., Cat. No. 400017) according to the protocol provided by the manufacturer. Subsequently, zymosan (Wako Pure Chemical Industries, Ltd., Cat. No. 263-01491) was prepared at a dose of 20 μg/mL using the dilution medium supplied with the CH50 Serum Complement Titer Test Kit. The serum sample was diluted 40-fold using the same dilution medium. Sheep red blood cells, zymosan, and the diluted serum sample were each mixed in a volume of 50 μL, and the mixture was incubated at 37°C overnight. The next day, the assay plate was centrifuged at 2000 rpm at room temperature for 10 minutes, and then the absorbance of the supernatant was measured at 405 nm. Table 20 shows the values for the samples, where 100% of the complement activity was determined in the serum of each individual on the day before administration.

[0122]

[0123] [Example 8. Pharmacological test (hemolysis suppression effect with single administration)]

(Obtaining siRNA-LNP)

LNPs encapsulating siRNA were prepared in the same manner as in Example 3, except that the siRNAs listed in Table 21 were used. The product quality evaluation results for the obtained LNPs are presented in Table 22.

[0124]

[0125]

[0126] (In vivo assessment)

LNPs encapsulating PBS or siRNA listed in Table 21 herein were intravenously administered to BALB/c mice (7-week-old males, n=4 per group) via the tail vein at 0.3, 1, and 3 mg/kg siRNA doses. Blood samples were collected under anesthesia on the day before administration (day -1 in Table 23) and at 7 days, 13 days, 20 days, and 27 days after administration. Blood samples taken on each collection day were placed in a blood separation tube containing clot activator (Immuno-Biological Laboratories Co, Ltd., Cat. No. 31203) and centrifuged at 3000 rpm for 15 minutes, and the serum supernatant was collected and stored at -80°C. Thereafter, the complement activity in the serum was quantitatively determined as follows. Specifically, sheep red blood cells at a concentration of 1.5 × 10 ⁸ cells/mL were prepared using the CH50 Serum Complement Titer Assay Kit (DENKA SEIKEN Co., Ltd., Cat. No. 400017) according to the protocol provided by the manufacturer. Subsequently, zymosan (Wako Pure Chemical Industries, Ltd., Cat. No. 263-01491) was prepared at a dose of 20 μg/mL using the dilution medium supplied with the CH50 Serum Complement Titer Assay Kit. The serum sample was diluted 40-fold using the same dilution medium. Sheep red blood cells, zymosan, and diluted serum sample were mixed, each in a volume of 50 μl, and the mixture was incubated at 37°C overnight. The next day, the assay plate was centrifuged at 2000 rpm at room temperature for 10 minutes, and then the absorbance of the supernatant was measured at 405 nm. Table 23 shows the values for the samples, where 100% of the complement activity in the serum was determined on the day before administration to each individual. The complement activity of 1 mouse in the 1 mg/kg dose group was excluded because it appeared to be an outlier. Therefore, the value for the 1 mg/kg dose group in Table 23 is shown as the average of 3 mice. The values for the PBS administration group, the 0.3 mg/kg dose group, and the 3 mg/kg dose group in Table 23 are shown as the average of 4 mice. The results are also presented in Fig. 3.

[0127]

[0128] [Example 9. Pharmacological test (hemolysis suppression effect when administered once every two weeks)]

(Obtaining siRNA-LNP)

LNPs encapsulating siRNA were prepared in the same manner as in Example 8.

[0129] (In vivo assessment)

LNPs encapsulating PBS or siRNA listed in Table 21 of Example 8 were administered intravenously to BALB/c mice (7-week-old males, n=4 per group) via the tail vein at 0.3, 1, and 3 mg/kg siRNA doses once every two weeks. Blood samples were collected under anesthesia the day before administration (day -1 in Table 24) and at 7 days, 13 days, 20 days, and 27 days after administration. Blood samples collected on each sample collection day were placed in a blood separation tube containing a coagulation activator (Immuno-Biological Laboratories Co, Ltd., Cat. No. 31203) and centrifuged at 3000 rpm for 15 minutes, and then the supernatant serum was collected and stored at -80°C. Thereafter, the complement activity in the serum was quantitatively determined as follows. Specifically, sheep red blood cells with a concentration of 1.5 × ¹⁰⁸ cells/mL were prepared using a CH50 Serum Complement Titer Assay Kit (DENKA SEIKEN Co., Ltd., Cat. No. 400017) according to the protocol provided by the manufacturer. Subsequently, zymosan (Wako Pure Chemical Industries, Ltd., Cat. No. 263-01491) was prepared at a dose of 20 μg/mL using the dilution medium supplied with the CH50 Serum Complement Titer Assay Kit. The serum sample was diluted 40-fold using the same dilution medium. Sheep red blood cells, zymosan, and the diluted serum sample were each mixed in a volume of 50 μL, and the mixture was incubated at 37°C overnight. The next day, the assay plate was centrifuged at 2000 rpm at room temperature for 10 minutes, and then the absorbance of the supernatant was measured at 405 nm. Table 24 shows the values for the samples, where 100% of the serum complement activity was determined on the day before administration to each individual. The results are also shown in Fig. 4.

[0130]

[0131] [Example 10. Pharmacological test (hemolysis suppression effect when administered once every two weeks)]

(Obtaining siRNA-LNP)

LNPs encapsulating siRNA were prepared in the same manner as in Example 8.

[0132] (In vivo assessment)

LNPs encapsulating PBS or siRNA listed in Table 21 in Example 8 were administered intravenously to BALB/c mice (7-week-old males, n=4 per group) via the tail vein at 0.3, 1, and 3 mg/kg siRNA doses once every two weeks. Blood samples were collected under anesthesia the day before administration (day -1 in Table 25) and at 7 days, 13 days, 20 days, 27 days, 34 days, 41 days, 48 days, and 55 days after administration. Blood samples collected on each sample collection day were placed in a blood separation tube containing a coagulation activator (Immuno-Biological Laboratories Co, Ltd., Cat. No. 31203) and centrifuged at 3000 rpm for 15 minutes, and then the supernatant serum was collected and stored at -80°C. Thereafter, the complement activity in the serum was quantitatively determined as follows. Specifically, sheep red blood cells with a concentration of 1.5 × ¹⁰⁸ cells/mL were prepared using a CH50 Serum Complement Titer Assay Kit (DENKA SEIKEN Co., Ltd., Cat. No. 400017) according to the protocol provided by the manufacturer. Subsequently, zymosan (Wako Pure Chemical Industries, Ltd., Cat. No. 263-01491) was prepared at a dose of 20 μg/mL using the dilution medium supplied with the CH50 Serum Complement Titer Assay Kit. The serum sample was diluted 40-fold using the same dilution medium. Sheep red blood cells, zymosan, and the diluted serum sample were each mixed in a volume of 50 μL, and the mixture was incubated at 37°C overnight. The next day, the assay plate was centrifuged at 2000 rpm at room temperature for 10 minutes, and then the absorbance of the supernatant was measured at 405 nm. Table 25 shows the values for the samples, where 100% of the serum complement activity was determined on the day before administration to each individual. The results are also shown in Fig. 5.

[0133]

[0134] [Example 11. Pharmacological test (hemolysis suppression effect when administered once every three weeks)]

(Obtaining siRNA-LNP)

LNPs encapsulating siRNA were prepared in the same manner as in Example 8.

[0135] (In vivo assessment)

LNPs encapsulating PBS or siRNA listed in Table 21 of Example 8 were administered intravenously to BALB/c mice (7-week-old males, n=4 per group) via the tail vein at 0.3, 1, and 3 mg/kg siRNA once every three weeks. Blood samples were collected under anesthesia one day before administration (day -1 in Table 26) and at 7 days, 13 days, 20 days, 27 days, 34 days, 41 days, 48 days, and 55 days after administration. Blood samples collected on each sample collection day were placed in a blood separation tube containing a coagulation activator (Immuno-Biological Laboratories Co, Ltd., Cat. No. 31203) and centrifuged at 3000 rpm for 15 minutes, and then the supernatant serum was collected and stored at -80°C. Thereafter, the complement activity in the serum was quantitatively determined as follows. Specifically, sheep red blood cells with a concentration of 1.5 × ¹⁰⁸ cells/mL were prepared using a CH50 Serum Complement Titer Assay Kit (DENKA SEIKEN Co., Ltd., Cat. No. 400017) according to the protocol provided by the manufacturer. Subsequently, zymosan (Wako Pure Chemical Industries, Ltd., Cat. No. 263-01491) was prepared at a dose of 20 μg/mL using the dilution medium supplied with the CH50 Serum Complement Titer Assay Kit. The serum sample was diluted 40-fold using the same dilution medium. Sheep red blood cells, zymosan, and the diluted serum sample were each mixed in a volume of 50 μL, and the mixture was incubated at 37°C overnight. The next day, the assay plate was centrifuged at 2000 rpm at room temperature for 10 minutes, and then the absorbance of the supernatant was measured at 405 nm. Table 26 shows the values for the samples, where 100% of the serum complement activity was determined on the day before administration to each individual. The results are also shown in Fig. 6.

[0136]

[0137] [Example 12. Physical Properties Test]

(Diameter of siRNA-LNP particles with changing pH of LNP solution)

siRNA-008-34 shown in Table 4 was dissolved in 10 mM sodium citrate (pH 4.0) to prepare a diluted siRNA solution. The lipid solution was prepared by dissolving 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate, DSPC (NIPPON FINE CHEMICAL CO., LTD.), cholesterol (Dishman Carbogen Amcis Limited), and MPEG2000-DMG (NOF CORPORATION) in ethanol at a molar ratio of 60/10.5/28/1.5. LNP was prepared by mixing the diluted siRNA solution and lipid solution at flow rates of 3:1. The obtained LNP solution was exchanged with PBS (pH 7.5) according to the conventional method, and then the LNP was concentrated. An aqueous solution of hydrochloric acid or sodium hydroxide was added to the LNP concentrate to adjust the pH to a range of 6.0 to 8.5. After adjusting the pH, the LNP was stored in a cool place.

[0138] The particle size measurement results of these LNPs using a particle size analyzer (Malvern Panalytical Ltd., Zetasizer Nano ZS) are shown in Table 27.

[0139]

[0140] [Example 13. Physical Properties Test]

(Diameter of siRNA-LNP particles with changing pH of LNP solution)

siRNA-008-34 shown in Table 4 was dissolved in 10 mM sodium citrate (pH 4.0) to prepare a diluted siRNA solution. The lipid solution was prepared by dissolving 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate, DSPC (NIPPON FINE CHEMICAL CO., LTD.), cholesterol (Dishman Carbogen Amcis Limited), and MPEG2000-DMG (NOF CORPORATION) in ethanol at a molar ratio of 60/10.5/28/1.5. LNP was prepared by mixing the diluted siRNA solution and lipid solution at flow rates of 3:1. The obtained LNP solution was exchanged with PBS (pH 7.5) according to the conventional method, and then the LNP was concentrated. An aqueous solution of hydrochloric acid or sodium hydroxide was added to the LNP concentrate to adjust the pH to a range of 6.5 to 8.5. After adjusting the pH, the LNP was stored in a cool place (5°C).

[0141] The particle size measurement results of these LNPs using a particle size analyzer (NICOMP380) are shown in Table 28. The appearance of the LNPs at each pH was a homogeneous solution with a characteristic opalescence of white or yellowish-white color, and no change was detected from the time immediately after pH adjustment to the time of storage for 1 month.

[0142]

[0143] [Example 14. Physical Properties Test]

(Diameter of siRNA-LNP particles with changing pH of LNP solution)

siRNA-008-34 shown in Table 4 was dissolved in 10 mM sodium citrate (pH 4.0) to prepare a diluted siRNA solution. The lipid solution was prepared by dissolving 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate, DSPC (NIPPON FINE CHEMICAL CO., LTD.), cholesterol (Dishman Carbogen Amcis Limited), and MPEG2000-DMG (NOF CORPORATION) in ethanol at a molar ratio of 60/10.5/28/1.5. LNP was prepared by mixing the diluted siRNA solution and lipid solution at flow rates of 3:1. The obtained LNP solution was exchanged with PBS (pH 7.5) according to the conventional method, and then the LNP was concentrated. An aqueous solution of hydrochloric acid or sodium hydroxide was added to the LNP concentrate to adjust the pH to a range of 6.5 to 8.5. After adjusting the pH, the LNP was stored in a cool place (5°C).

[0144] The particle size measurement results of the LNP data using a particle size analyzer (NICOMP380) are shown in Table 29.

[0145]

[0146] [Example 15. Physical Properties Test]

(Diameter of siRNA-LNP particles with changing pH of LNP solution)

siRNA-008-34 shown in Table 4 was dissolved in 10 mM sodium citrate (pH 4.0) to prepare a diluted siRNA solution. The lipid solution was prepared by dissolving 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate, DSPC (NIPPON FINE CHEMICAL CO., LTD.), cholesterol (Dishman Carbogen Amcis Limited), and MPEG2000-DMG (NOF CORPORATION) in ethanol at a molar ratio of 60/10.5/28/1.5. LNP was prepared by mixing the diluted siRNA solution and lipid solution at flow rates of 3:1. The obtained LNP solution was exchanged with PBS (pH 7.5) according to the conventional method, and then the LNP was concentrated. An aqueous solution of hydrochloric acid or sodium hydroxide was added to the LNP concentrate to adjust the pH to a range of 6.5 to 8.5. After pH adjustment, the LNP was stored at room temperature (25°C).

[0147] The particle size measurement results of the LNP data using a particle size analyzer (NICOMP380) are shown in Table 30.

[0148]

[0149] [Example 16. Physical Properties Test]

(Diameter of siRNA-LNP particles with changing pH of LNP solution)

siRNA-008-34 shown in Table 4 was dissolved in 10 mM sodium citrate (pH 4.0) to obtain a diluted siRNA solution. The lipid solution was prepared by dissolving 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate, DSPC (Lipoid GmbH), cholesterol (Dishman Carbogen Amcis Limited), and MPEG2000-DMG (NOF CORPORATION) in ethanol at a molar ratio of 60/10.5/28/1.5. LNP was prepared by mixing the diluted siRNA solution and lipid solution at flow rates of 3:1. The obtained LNP solution was exchanged with PBS (pH 7.7) according to a conventional method, and then the LNP was concentrated. Subsequently, the LNP concentrate was clarified by filtration and adjusted in concentration, followed by sterilization by filtration. The resulting LNP solution was applied to a dialysis membrane and dialyzed with a Britton-Robinson (BR) buffer solution at pH 2.0/5.0/9.0/10.0/11.0 at room temperature to obtain LNP solutions with different pH. After completion of dialysis, the pH of each LNP solution was confirmed, and the LNP solution was defined as "immediately after pH adjustment". After pH adjustment, the LNP solutions were stored in a cool place (5°C).

[0150] The particle size measurement results of the LNP data using a particle size analyzer (NICOMP380) are shown in Table 31.

[0151]

--->

SEQUENCE LIST

<110> Eisai R&D Management Co., Ltd.

<120> PHARMACEUTICAL COMPOSITION CONTAINING DOUBLE-STRANTED RIBONUCLEIIC ACID CAPABLE OF SUPPRESSION OF COMPLEMENT COMPONENT C5

<130> FP20-0635-00

<150> JP2019-236918

<151> 2019-12-26

<160> 172

<170> PatentIn version 3.5

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<222> (11)..(11)

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<222> (18)..(18)

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<222> (20)..(21)

<223> n=deoxythymidine

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<222> (20)..(21)

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<221> other_feature

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<223> n=deoxythymidine

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<221> other_feature

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<221> other_feature

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<223> n=deoxythymidine

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<221> other_feature

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uuucuaauuc caaucacagn n 21

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<222> (20)..(21)

<223> n=deoxythymidine

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<221> other_feature

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<223> phosphorothioate bond

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aaggcaaagg uguucaaagn n 21

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<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (8)..(8)

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<220>

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<223> n=deoxythymidine

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cuuugaacac cuuugccuun n 21

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<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(14)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> cm

<220>

<221> other_feature

<222> (19)..(19)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 11

gaaaggaacu guuuacaacn n 21

<210> 12

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 12

guuguaaaca guuccuuucn n 21

<210> 13

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 13

uggguauaugu guugcugaun n 21

<210> 14

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 14

aucagcaaca caauauaccan n 21

<210> 15

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> cm

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (7)..(8)

<223> µm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (12)..(14)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<220>

<221> other_feature

<222> (17)..(17)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 15

acugucuuaa cuuucauagn n 21

<210> 16

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (11)..(11)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 16

cuaugaaagu uaagacagun n 21

<210> 17

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (4)..(5)

<223> cm

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (15)..(17)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 17

gugccagcua caagcccagn n 21

<210> 18

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 18

cugggcuugu agcuggcacn n 21

<210> 19

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (7)..(7)

<223> cm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(12)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (18)..(18)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 19

aaggaacugu uuacaacuan n 21

<210> 20

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 20

uaguuguaaa caguuccuun n 21

<210> 21

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (2)..(3)

<223> cm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (16)..(17)

<223> cm

<220>

<221> other_feature

<222> (18)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 21

uccucuggaa auuggccuun n 21

<210> 22

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 22

aaggccaauu uccagaggan n 21

<210> 23

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(2)

<223> µm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (12)..(12)

<223> µm

<220>

<221> other_feature

<222> (14)..(16)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 23

uugaaaggaa cuguuuacan n 21

<210> 24

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> cm

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 24

uguaaacagu uccuuucaan n 21

<210> 25

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (11)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 25

aaaggaacug uuuacaacun n 21

<210> 26

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 26

aguuguaaac aguuccuuun n 21

<210> 27

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (9)..(11)

<223> µm

<220>

<221> other_feature

<222> (13)..(13)

<223> cm

<220>

<221> other_feature

<222> (16)..(16)

<223> cm

<220>

<221> other_feature

<222> (17)..(17)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 27

aggaacuguu uacaacuaun n 21

<210> 28

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (12)..(12)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 28

auaguuguaa acaguuccun n 21

<210> 29

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 29

uacacugaag cauuugaugn n 21

<210> 30

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> cm

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> cm

<220>

<221> other_feature

<222> (18)..(18)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 30

caucaaugc uucaguguan n 21

<210> 31

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> cm

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(13)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 31

cacugaagca uuugaugcan n 21

<210> 32

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 32

ugcaucaaau gcuucagugn n 21

<210> 33

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> cm

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> cm

<220>

<221> other_feature

<222> (9)..(11)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> cm

<220>

<221> other_feature

<222> (19)..(19)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 33

cugaagcauu ugaugcaacn n 21

<210> 34

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 34

guugcaucaa augcuucagn n 21

<210> 35

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(2)

<223> µm

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (14)..(15)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> cm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 35

uucugcaacu gaauucgaun n 21

<210> 36

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 36

aucgaauuca guugcagaan n 21

<210> 37

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> µm

<220>

<221> other_feature

<222> (13)..(15)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 37

ugaaaggaac uguuuacaan n 21

<210> 38

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 38

uuguaaacag uuccuuucan n 21

<210> 39

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> cm

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<220>

<221> other_feature

<222> (10)..(12)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 39

acugaagcau uugaugcaan n 21

<210> 40

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (7)..(7)

<223> cm

<220>

<221> other_feature

<222> (16)..(16)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 40

uugcaucaaa ugcuucagun n 21

<210> 41

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> cm

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (13)..(14)

<223> cm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (17)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 41

cauacagaca aaccuguuun n 21

<210> 42

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 42

aaacagguuu gucuguaugn n 21

<210> 43

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (7)..(7)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> µm

<220>

<221> other_feature

<222> (13)..(14)

<223> cm

<220>

<221> other_feature

<222> (15)..(17)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 43

aaacaacaag uaccuuuaun n 21

<210> 44

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 44

auaaagguac uuguuguuun n 21

<210> 45

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<220>

<221> other_feature

<222> (12)..(13)

<223> cm

<220>

<221> other_feature

<222> (14)..(14)

<223> µm

<220>

<221> other_feature

<222> (16)..(18)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 45

auacagacaa accuguuuan n 21

<210> 46

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (17)..(17)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 46

uaaacagguu ugucuguaun n 21

<210> 47

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (11)..(12)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 47

gguauaugug uugcugauan n 21

<210> 48

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (7)..(7)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> cm

<220>

<221> other_feature

<222> (12)..(12)

<223> cm

<220>

<221> other_feature

<222> (14)..(14)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 48

uaucagcaac acauauaccn n 21

<210> 49

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (2)..(2)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> µm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 49

ucagaaaguc ugugaaggan n 21

<210> 50

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 50

uccuucacag acuuucugan n 21

<210> 51

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (2)..(2)

<223> cm

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (4)..(5)

<223> cm

<220>

<221> other_feature

<222> (9)..(10)

<223> cm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 51

ucuccaggcc aaacugugun n 21

<210> 52

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> cm

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 52

acacaguuug gccuggagan n 21

<210> 53

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> cm

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (11)..(12)

<223> cm

<220>

<221> other_feature

<222> (13)..(15)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 53

acaacaagua ccuuuauaun n 21

<210> 54

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 54

auauaaaggu acuuguugun n 21

<210> 55

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> cm

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(11)

<223> cm

<220>

<221> other_feature

<222> (12)..(14)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (18)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 55

caacaaguac cuuuauauun n 21

<210> 56

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (11)..(11)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 56

aauauaaagg uacuuguugn n 21

<210> 57

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(3)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (6)..(7)

<223> cm

<220>

<221> other_feature

<222> (11)..(12)

<223> cm

<220>

<221> other_feature

<222> (16)..(16)

<223> cm

<220>

<221> other_feature

<222> (17)..(17)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 57

auucuccagg ccaaacugun n 21

<210> 58

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 58

acaguuuggc cuggagaaun n 21

<210> 59

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (8)..(9)

<223> cm

<220>

<221> other_feature

<222> (12)..(12)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (14)..(15)

<223> cm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 59

guggcaacca gcuccaggun n 21

<210> 60

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 60

accuggagcu gguugccacn n 21

<210> 61

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (7)..(7)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<220>

<221> other_feature

<222> (15)..(16)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 61

aagagacauc ugacuuggan n 21

<210> 62

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 62

uccaagucag augucucuun n 21

<210> 63

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(3)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> µm

<220>

<221> other_feature

<222> (15)..(16)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 63

auucugcaac ugaauucgan n 21

<210> 64

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 64

ucgaauucag uugcagaaun n 21

<210> 65

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(2)

<223> µm

<220>

<221> other_feature

<222> (3)..(4)

<223> cm

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (13)..(14)

<223> µm

<220>

<221> other_feature

<222> (17)..(18)

<223> cm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 65

uuccugga aauuggccun n 21

<210> 66

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (12)..(12)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 66

aggccaauuu ccagaggaan n 21

<210> 67

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> cm

<220>

<221> other_feature

<222> (14)..(16)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 67

aacaacaagu accuuuauan n 21

<210> 68

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 68

uauaaaggua cuuguuguun n 21

<210> 69

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (8)..(9)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (12)..(12)

<223> µm

<220>

<221> other_feature

<222> (13)..(13)

<223> cm

<220>

<221> other_feature

<222> (14)..(14)

<223> µm

<220>

<221> other_feature

<222> (15)..(17)

<223> cm

<220>

<221> other_feature

<222> (18)..(18)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 69

aauauguccu cucucccuan n 21

<210> 70

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 70

uagggagaga ggacauauun n 21

<210> 71

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> cm

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<220>

<221> other_feature

<222> (16)..(17)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 71

acucacuaua auuacuugan n 21

<210> 72

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(12)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 72

ucaaguaauu auagugagun n 21

<210> 73

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(12)

<223> µm

<220>

<221> other_feature

<222> (15)..(16)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 73

auaacucacu auaauuacun n 21

<210> 74

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 74

aguaauuaua gugaguuaun n 21

<210> 75

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (9)..(10)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> µm

<220>

<221> other_feature

<222> (12)..(12)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (16)..(18)

<223> cm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 75

aaauaugucc ucucucccun n 21

<210> 76

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (13)..(13)

<223> cm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 76

agggagagag gacauauuun n 21

<210> 77

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (7)..(11)

<223> µm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 77

aagauauuuu uauaauaaan n 21

<210> 78

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 78

uuuauuauaaaaauaucuun n 21

<210> 79

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> cm

<220>

<221> other_feature

<222> (12)..(12)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 79

aaaauaacuc acuauaauun n 21

<210> 80

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 80

aauuauagug aguuauuuun n 21

<210> 81

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> cm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (12)..(12)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> µm

<220>

<221> other_feature

<222> (17)..(18)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 81

aaauaacuca cuauaauuan n 21

<210> 82

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 82

uaauuauagu gaguuauuun n 21

<210> 83

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (4)..(5)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(12)

<223> µm

<220>

<221> other_feature

<222> (13)..(13)

<223> cm

<220>

<221> other_feature

<222> (14)..(14)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> cm

<220>

<221> other_feature

<222> (17)..(17)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 83

guguuaaaau gucugcugun n 21

<210> 84

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> cm

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (14)..(14)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 84

acagcagaca uuuuaacacn n 21

<210> 85

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (7)..(11)

<223> µm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<220>

<221> other_feature

<222> (18)..(18)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 85

aaaauguuuu ugucaaguan n 21

<210> 86

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 86

uacuugacaa aaacauuuun n 21

<210> 87

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> cm

<220>

<221> other_feature

<222> (2)..(3)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (7)..(7)

<223> cm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (12)..(12)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<220>

<221> other_feature

<222> (15)..(16)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 87

cuuacgcuga guacuucgan n 21

<210> 88

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 88

ucgaaguacu cagcguaagn n 21

<210> 89

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 89

aggcaaaggu guucaaagan n 21

<210> 90

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 90

ucuuugaaca ccuuugccun n 21

<210> 91

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<400> 91

aggcaaaggu guucaaaga 19

<210> 92

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<400> 92

ucuuugaaca ccuuugccu 19

<210> 93

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(2)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (10)..(21)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<400> 93

aggcaaaggu guucaaagau u 21

<210> 94

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(2)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 94

ucuuugaaca ccuuugccuu u 21

<210> 95

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(2)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (2)..(3)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 95

aggcaaaggu guucaaagau u 21

<210> 96

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(3)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 96

ucuuugaaca ccuuugccuu u 21

<210> 97

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(2)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<220>

<221> other_feature

<222> (19)..(20)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 97

aggcaaaggu guucaaagau u 21

<210> 98

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(2)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (19)..(21)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 98

ucuuugaaca ccuuugccuu u 21

<210> 99

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<400> 99

aggcaaaggu guucaaaga 19

<210> 100

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 100

ucuuugaaca ccuuugccuu u 21

<210> 101

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (12)..(12)

<223> µm

<220>

<221> other_feature

<222> (14)..(15)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> cm

<400> 101

auaggcaaag guguucaaag a 21

<210> 102

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 102

ucuuugaaca ccuuugccuu u 21

<210> 103

<400> 103

000

<210> 104

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 104

ucuuugaaca ccuuugccuu u 21

<210> 105

<400> 105

000

<210> 106

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 106

cuuugaacac cuuugccuun n 21

<210> 107

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> µm

<220>

<221> other_feature

<222> (13)..(14)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<400> 107

aaggcaaagg uguucaaag 19

<210> 108

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<400> 108

cuuugaacac cuuugccuu 19

<210> 109

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(14)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> cm

<220>

<221> other_feature

<222> (19)..(19)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 109

gaaaggaacu guuuacaacn n 21

<210> 110

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 110

guuguaaaca guuccuuucn n 21

<210> 111

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (13)..(15)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (19)..(19)

<223> cm

<400> 111

gaaaggaacu guuuacaac 19

<210> 112

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<400> 112

guuguaaaca guuccuuuc 19

<210> 113

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 113

ugguauaugu guugcugaun n 21

<210> 114

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 114

aucagcaaca caauauaccan n 21

<210> 115

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<400> 115

ggguauaugu guugcugau 19

<210> 116

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<400> 116

aucagcaaca cauauaacca 19

<210> 117

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (1)..(2)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(21)

<223> µm

<400> 117

uggguauaugu guugcugauu u 21

<210> 118

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(2)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 118

aucagcaaca caauauaccau u 21

<210> 119

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(3)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(21)

<223> µm

<400> 119

uggguauaugu guugcugauu u 21

<210> 120

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(3)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 120

aucagcaaca caauauaccau u 21

<210> 121

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (1)..(2)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(21)

<223> µm

<220>

<221> other_feature

<222> (19)..(21)

<223> phosphorothioate bond

<400> 121

uggguauaugu guugcugauu u 21

<210> 122

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(2)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (19)..(21)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 122

aucagcaaca caauauaccau u 21

<210> 123

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<400> 123

ggguauaugu guugcugau 19

<210> 124

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 124

aucagcaaca caauauaccau u 21

<210> 125

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(3)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(12)

<223> µm

<220>

<221> other_feature

<222> (14)..(15)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (18)..(18)

<223> µm

<220>

<221> other_feature

<222> (21)..(21)

<223> µm

<400> 125

auugguauau guguugcuga u 21

<210> 126

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 126

aucagcaaca caauauaccau u 21

<210> 127

<211> 22

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (3)..(4)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (11)..(11)

<223> µm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(16)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (22)..(22)

<223> µm

<400> 127

uauugguaua uguguugcug au 22

<210> 128

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 128

aucagcaaca caauauaccau u 21

<210> 129

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (3)..(4)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (11)..(11)

<223> µm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(16)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (22)..(22)

<223> µm

<220>

<221> other_feature

<222> (23)..(23)

<223> n=inverted deoxythymidine

<400> 129

uauugguaua uguguugcug aun 23

<210> 130

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 130

aucagcaaca caauauaccau u 21

<210> 131

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(21)

<223> µm

<400> 131

uggguauaugu guugcugauu u 21

<210> 132

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 132

aucagcaaca caauauaccau u 21

<210> 133

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<220>

<221> other_feature

<222> (19)..(21)

<223> µm

<400> 133

uggguauaugu guugcugauu u 21

<210> 134

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 134

aucagcaaca caauauaccau u 21

<210> 135

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(3)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(12)

<223> µm

<220>

<221> other_feature

<222> (14)..(15)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (18)..(18)

<223> µm

<400> 135

auugguauau guguugcuga 20

<210> 136

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> cm

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> cm

<220>

<221> other_feature

<222> (12)..(12)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (19)..(20)

<223> µm

<400> 136

ucagcaacac auauaccauu 20

<210> 137

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (11)..(11)

<223> µm

<220>

<221> other_feature

<222> (13)..(14)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> cm

<220>

<221> other_feature

<222> (17)..(17)

<223> µm

<220>

<221> other_feature

<222> (20)..(20)

<223> µm

<400> 137

augguauaug uguugcugau 20

<210> 138

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (19)..(20)

<223> µm

<400> 138

aucagcaaca caauauaccuu 20

<210> 139

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<400> 139

ggguauaugu guugcugau 19

<210> 140

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(23)

<223> µm

<400> 140

aucagcaaca cauauaccau uuu 23

<210> 141

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<400> 141

ggguauaugu guugcugau 19

<210> 142

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(23)

<223> µm

<400> 142

aucagcaaca cauauaccau uuu 23

<210> 143

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<400> 143

ggguauaugu guugcugau 19

<210> 144

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<220>

<221> other_feature

<222> (22)..(23)

<223> 2'-O-methyladenosine

<400> 144

aucagcaaca cauauaccau uaa 23

<210> 145

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<400> 145

ggguauaugu guugcugau 19

<210> 146

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<220>

<221> other_feature

<222> (21)..(23)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (22)..(23)

<223> 2'-O-methyladenosine

<400> 146

aucagcaaca cauauaccau uaa 23

<210> 147

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<400> 147

ggguauaugu guugcugau 19

<210> 148

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (19)..(21)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<220>

<221> other_feature

<222> (22)..(23)

<223> 2'-O-methyladenosine

<400> 148

aucagcaaca cauauaccau uaa 23

<210> 149

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<400> 149

ggguauaugu guugcugau 19

<210> 150

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> 2'-O-methyladenosine

<220>

<221> other_feature

<222> (1)..(2)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (19)..(21)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<220>

<221> other_feature

<222> (22)..(23)

<223> 2'-O-methyladenosine

<400> 150

aucagcaaca cauauaccau uaa 23

<210> 151

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<400> 151

ggguauaugu guugcugau 19

<210> 152

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> 2'-O-methyladenosine

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(23)

<223> µm

<400> 152

aucagcaaca cauauaccau uuu 23

<210> 153

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (1)..(2)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<400> 153

ggguauaugu guugcugau 19

<210> 154

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (19)..(21)

<223> phosphorothioate bond

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<220>

<221> other_feature

<222> (22)..(23)

<223> 2'-O-methyladenosine

<400> 154

aucagcaaca cauauaccau uaa 23

<210> 155

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(12)

<223> µm

<220>

<221> other_feature

<222> (15)..(16)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 155

auaacucacu auaauuacun n 21

<210> 156

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 156

aguaauuaua gugaguuaun n 21

<210> 157

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(12)

<223> µm

<220>

<221> other_feature

<222> (15)..(16)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (19)..(19)

<223> µm

<400> 157

auaacucacu auaauuacu 19

<210> 158

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> µm

<400> 158

aguaauuaua gugaguuau 19

<210> 159

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> cm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (19)..(21)

<223> µm

<400> 159

uggguauaugu guugcugauu u 21

<210> 160

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> cm

<220>

<221> other_feature

<222> (6)..(6)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 160

aucagcaaca caauauaccau u 21

<210> 161

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(13)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 161

aggcaaaggu guucaaagau u 21

<210> 162

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 162

ucuuugaaca ccuuugccuu u 21

<210> 163

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(14)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> cm

<220>

<221> other_feature

<222> (19)..(19)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 163

gaaaggaacu guuuacaacu u 21

<210> 164

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> µm

<400> 164

guuguaaaca guuccuuucu u 21

<210> 165

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> cm

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(10)

<223> µm

<220>

<221> other_feature

<222> (12)..(12)

<223> µm

<220>

<221> other_feature

<222> (14)..(15)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (18)..(18)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 165

acugguauau guguugcugn n 21

<210> 166

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> cm

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (7)..(7)

<223> cm

<220>

<221> other_feature

<222> (9)..(9)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> µm

<220>

<221> other_feature

<222> (13)..(13)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 166

cagcaacaca uauaccagun n 21

<210> 167

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> cm

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (11)..(11)

<223> µm

<220>

<221> other_feature

<222> (13)..(14)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> cm

<220>

<221> other_feature

<222> (17)..(17)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 167

cugguauaug uguugcugan n 21

<210> 168

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> cm

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> cm

<220>

<221> other_feature

<222> (12)..(12)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 168

ucagcaacac auauaccagn n 21

<210> 169

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (3)..(3)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> µm

<220>

<221> other_feature

<222> (7)..(7)

<223> µm

<220>

<221> other_feature

<222> (9)..(9)

<223> µm

<220>

<221> other_feature

<222> (11)..(12)

<223> µm

<220>

<221> other_feature

<222> (14)..(14)

<223> cm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (18)..(18)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 169

gguauaugug uugcugauan n 21

<210> 170

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (1)..(1)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> cm

<220>

<221> other_feature

<222> (7)..(7)

<223> cm

<220>

<221> other_feature

<222> (10)..(10)

<223> cm

<220>

<221> other_feature

<222> (12)..(12)

<223> cm

<220>

<221> other_feature

<222> (14)..(14)

<223> µm

<220>

<221> other_feature

<222> (16)..(16)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 170

uaucagcaac acauauaccn n 21

<210> 171

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> synthesized sense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (4)..(4)

<223> µm

<220>

<221> other_feature

<222> (6)..(6)

<223> µm

<220>

<221> other_feature

<222> (8)..(8)

<223> µm

<220>

<221> other_feature

<222> (10)..(11)

<223> µm

<220>

<221> other_feature

<222> (13)..(13)

<223> cm

<220>

<221> other_feature

<222> (14)..(14)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> µm

<220>

<221> other_feature

<222> (19)..(19)

<223> cm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 171

guauaugugu ugcugauacn n 21

<210> 172

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthesized antisense siRNA sequence

<220>

<221> other_feature

<222> (2)..(2)

<223> µm

<220>

<221> other_feature

<222> (5)..(5)

<223> cm

<220>

<221> other_feature

<222> (8)..(8)

<223> cm

<220>

<221> other_feature

<222> (11)..(11)

<223> cm

<220>

<221> other_feature

<222> (13)..(13)

<223> cm

<220>

<221> other_feature

<222> (15)..(15)

<223> µm

<220>

<221> other_feature

<222> (17)..(17)

<223> µm

<220>

<221> other_feature

<222> (20)..(21)

<223> n=deoxythymidine

<220>

<221> other_feature

<222> (20)..(21)

<223> phosphorothioate bond

<400> 172

guaucagcaa cacaauauacn n 21

<---

Claims (35)

1. A pharmaceutical composition for inhibiting the expression of complement component C5, comprising lipid complex, wherein the lipid complex comprises a double-stranded ribonucleic acid comprising a sense strand consisting of the nucleotide sequence presented under SEQ ID NO: 145 and an antisense strand consisting of the nucleotide sequence presented under SEQ ID NO: 146, and The pH of the lipid complex solution is 2.0 or greater and 5.0 or less, or 7.5 or greater and 11.0 or less, and pharmaceutically acceptable carrier. 2. The pharmaceutical composition according to claim 1, wherein the pH of the lipid complex solution is 7.5 or greater and 10.0 or less. 3. A pharmaceutical composition according to any one of claims 1, 2, wherein the average particle size of the lipid complex is 100 nm or less. 4. The pharmaceutical composition according to any one of claims 1, 2, wherein the average particle size of the lipid complex is 65 nm or more and 100 nm or less. 5. The pharmaceutical composition according to any one of claims 1, 2, wherein the average particle size of the lipid complex is 80 nm or more and 100 nm or less. 6. The pharmaceutical composition according to any one of claims 1-5, wherein the change in the average particle size of the lipid complex after storage for 2 weeks is 10% or less of the average particle size of the lipid complex before storage. 7. The pharmaceutical composition according to claim 6, wherein the change in the average particle size of the lipid complex is an increase in the average particle size of the lipid complex. 8. A pharmaceutical composition according to any one of claims 1-7, wherein the lipid complex comprises cationic lipid and at least one lipid selected from the group consisting of a neutral lipid, a polyethyleneglycol-modified lipid, and a sterol. 9. The pharmaceutical composition of claim 8, wherein the cationic lipid is 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl-1-methylpiperidine-4-carboxylate. 10. The pharmaceutical composition according to any one of claims 1-9, wherein the lipid complex is a lipid nanoparticle (LNP). 11. A pharmaceutical composition according to any one of claims 1-10, wherein the lipid complex encapsulates a double-stranded ribonucleic acid containing a combination of a sense strand and an antisense strand. 12. A pharmaceutical composition according to any one of claims 1-11 for use in the treatment of paroxysmal nocturnal hemoglobinuria. 13. A pharmaceutical composition according to any one of claims 1-11 for use in the treatment of atypical hemolytic uremic syndrome. 14. A method for producing a pharmaceutical composition according to any one of paragraphs 1-13, comprising mixing an organic solvent comprising (I) a cationic lipid and (II) at least one lipid selected from the group consisting of a neutral lipid, a polyethyleneglycol-modified lipid, and a sterol, and an aqueous solution comprising a double-stranded ribonucleic acid comprising a combination of a sense strand comprising a nucleotide sequence as set forth in SEQ ID NO: 145 and an antisense strand comprising a nucleotide sequence as set forth in SEQ ID NO: 146, to obtain a lipid complex solution; adjusting the pH of the lipid complex solution to 2.0 or greater and 5.0 or less, or 7.5 or greater and 11 or less. 15. The method according to claim 14 for obtaining a pharmaceutical composition according to any one of claims 1-13, comprising bringing the pH of the lipid complex solution to 7.5 or greater and 10.0 or less. 16. A method for stabilizing a pharmaceutical composition according to any one of paragraphs 1-13, comprising mixing an organic solvent comprising (I) a cationic lipid and (II) at least one lipid selected from the group consisting of a neutral lipid, a polyethyleneglycol-modified lipid, and a sterol, and an aqueous solution comprising a double-stranded ribonucleic acid comprising a combination of a sense strand comprising a nucleotide sequence as set forth in SEQ ID NO: 145 and an antisense strand comprising a nucleotide sequence as set forth in SEQ ID NO: 146, to obtain a lipid complex solution; adjusting the pH of the lipid complex solution to 2.0 or greater and 5.0 or less, or 7.5 or greater and 11.0 or less. 17. The method according to claim 16 for stabilizing the pharmaceutical composition according to any one of claims 1-13, comprising bringing the pH of the lipid complex solution to 7.5 or greater and 10.0 or less. 18. The method according to claim 16 or 17 for stabilizing the pharmaceutical composition according to any one of claims 1-13, wherein the method of stabilizing the pharmaceutical composition is a method of suppressing a change in the average particle size of a lipid complex in the pharmaceutical composition. 19. The method of claim 18 for stabilizing the pharmaceutical composition of any one of claims 1-13, wherein the method of suppressing a change in the average particle size is a method of suppressing an increase in the average particle size. 20. A method for treating paroxysmal nocturnal hemoglobinuria, comprising administering a pharmaceutical composition according to any one of claims 1-11 to a patient in need thereof. 21. A method for treating atypical hemolytic uremic syndrome, comprising administering a pharmaceutical composition according to any of paragraphs 1-11 to a patient in need thereof. 22. Use of a lipid complex comprising a double-stranded ribonucleic acid comprising a sense strand consisting of a nucleotide sequence presented under SEQ ID NO: 145 and an antisense strand consisting of a nucleotide sequence presented under SEQ ID NO: 146 in the manufacture of a medicinal product comprising a pharmaceutical composition according to any one of claims 1-11 for the treatment of paroxysmal nocturnal hemoglobinuria. 23. Use of a lipid complex comprising a double-stranded ribonucleic acid comprising a sense strand consisting of a nucleotide sequence presented under SEQ ID NO: 145 and an antisense strand consisting of a nucleotide sequence presented under SEQ ID NO: 146 in the manufacture of a medicinal product comprising a pharmaceutical composition according to any one of claims 1-11 for the treatment of atypical hemolytic uremic syndrome.

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